Non-linear multiblock copolymer-drug conjugates for the delivery of active agents

ABSTRACT

Non-linear multiblock copolymer-drug conjugates for the treatment and prevention of diseases and disorders of the eye are provided. The polymer-drug conjugates can form nanoparticles, microparticles, and implants that are capable of effectively delivering therapeutic levels of one or more active agents for an extended period of time. Administration to the eye of an active agent in the form of a non-linear multiblock copolymer-drug conjugate produces decreased side effects when compared to administration of the active agent alone. Also provided are methods of treating intraocular neovascular diseases, such as wet age-related macular degeneration as well as diseases and disorders of the eye associated with inflammation, such as uveitis.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.16/182,261, filed Nov. 6, 2018, which is a continuation of U.S.application Ser. No. 13/797,531, filed Mar. 12, 2013, now U.S. Pat. No.10,159,743, issued Dec. 25, 2018, which claims benefit of and priorityto U.S. Provisional Application No. 61/611,975 filed Mar. 16, 2012,which are herein incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under CA140746,EY001765, CA151838, and EY012609 awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to non-linear multiblock copolymer-drugconjugates with improved efficacy, stability, safety and ease offormation into nano- and microparticles, as well as methods of usethereof for the controlled delivery of active agents, particularly forthe controlled delivery of active agents to the eye.

BACKGROUND OF THE INVENTION

Approximately 1.7 million Americans over the age of 65 suffer fromage-related macular degeneration (AMD). As the nation's populationcontinues to age, this number is expected to grow by an estimated200,000 new cases per year. Severe vision loss from AMD and otherdiseases affecting the posterior segment, including diabeticretinopathy, glaucoma, and retinitis pigmentosa accounts for most casesof irreversible blindness worldwide.

Currently, the treatment of posterior segment diseases is to asignificant extent limited by the difficulty in delivering effectivedoses of drugs to target tissues in the posterior eye while avoidingtoxicity. Four modes of administration are commonly used to deliverdrugs to the posterior segment of the eye: topical, systemic,intraocular, and periocular administration.

Topical administration, for example the application of solutions to thesurface of the eye, is the most common mode of administration oftherapeutics for the pharmacologic management of ocular disease. Topicaladministration has the advantage of being minimally invasive; however,many factors can limit its usefulness. Examples include the significantbarrier to solute flux provided by the corneal epithelium, and the rapidand extensive precorneal loss that occurs as the result of drainage andtear fluid turnover. It has been estimated that typically less than 5%of a topically applied drug permeates the cornea and reaches intraoculartissues. The major portion of the instilled dose is absorbedsystemically by way of the conjunctiva, through the highly vascularconjunctival stroma and through the lid margin vessels. Significantsystemic absorption also occurs when the solution enters thenasolacrimal duct and is absorbed by the nasal and nasopharyngealmucosa. Despite the relatively small proportion of a topically applieddrug dose that ultimately reaches anterior segment ocular tissues,topical formulations can be effective in some circumstances, largelybecause of the very high concentrations of drugs that can beadministered.

Recent advances in topical drug delivery have focused on improvingocular drug contact time and drug delivery from the surface of the eyeto the posterior segment. For example, ointments, gels, liposomeformulations, and various sustained and controlled-release substrates,such as the Ocusert® system, collagen shields, and hydrogel lenses, havebeen developed to improve ocular drug contact time. Topical deliverysystems using polymeric gels, colloidal systems, and cyclodextrins havealso been investigated in an effort to improve drug delivery to theposterior segment. In spite of these efforts, the delivery oftherapeutic doses of drugs to the posterior segment of the eye bytopical routes remains a significant challenge.

Drugs for the treatment of posterior segment diseases can also beadministered systemically. Although systemic administration can deliverdrugs to the posterior eye, large systemic doses are typically requiredto yield therapeutic drug levels in the posterior vitreous, retina, orchoroid. As a result, systemic administration is generally plagued bysignificant side effects associated with the administration of largesystemic doses of the therapeutic agent.

Periocular drug delivery using subconjunctival or retrobulbal injectionsor placement of sustained-release devices provides another route fordelivering drugs to the posterior tissues of the eye. This approachoffers the potential for localized, sustained-release drug delivery. Theaverage 17 cm² surface area of the human sclera accounts for 95% of thetotal surface area of the globe and provides a significantly largeravenue for drug diffusion to the inside of the eye than the 1-cm²surface area of the cornea. Also, regional differences in scleralthickness could be used to further optimize transscleral drug diffusionif sustained-release delivery devices or systems could be placed inregions where scleral permeability was greatest. The sclera, forexample, is 1.0 mm thick near the optic nerve and an average of 0.53 mmthick at the corneoscleral limbus and thins to an average of 0.39 mm atthe equator, where it can be as thin as 0.1 mm in a significant numberof eyes. See Geroski, et al. Invest. Ophthalmol. Vis. Sci. 41(5):961-964(2000).

Intravitreal injection represents the most common method foradministering therapeutic drug levels to the posterior segment of theeye. While intravitreal injection offers the opportunity to controlinitial drug levels in the posterior segment of the eye while minimizingany systemic toxicity associated with the drug, intravitrealadministration suffers some significant drawbacks. Intravitrealinjections have several inherent potential side effects, including arisk of retinal detachment, hemorrhage, endophthalmitis, and cataractdevelopment. Repeat injections are frequently required, and they are notalways well tolerated by the patient. Further, drugs injected directlyinto the vitreous are rapidly eliminated, making it difficult tomaintain therapeutically effective levels of the drug in the posteriorsegment.

For drugs that are administered to regions of the body where they arerapidly eliminated (e.g., the posterior segment of the eye), are used totreat chronic diseases or disorders, and/or have a narrowtherapeutically effective concentration range (i.e., therapeuticwindow), conventional drug delivery methods are inappropriate.Conventional drug administration involves periodic dosing of atherapeutic agent in a dosage formulation that ensures drug stability,activity, and bioavailability. Administration of the therapeutic agenttypically results in a sharp initial increase in drug concentration(often to toxic levels), followed by a steady decline in concentrationas the drug is cleared and/or metabolized. To maintain an effectiveconcentration of the therapeutic agent in the posterior segment for thetreatment of chronic eye diseases, repeated administration of the dosageformulation is typically required. The periodic drug delivery generatesa drug concentration profile that oscillates over time, often spiking totoxic levels and/or dipping below the therapeutic window.

Controlled release formulations offer the potential to improve patientoutcomes in these instances. Controlled release formulations provide theability to minimize/eliminate spikes in drug concentration, minimizingside effects and/or toxicity. Controlled release formulations can alsomaintain the drug concentration within the therapeutic window for longerperiods of time. As a result, these formulations are more comfortableand convenient for the patient, due to a diminished frequency of ocularinjections.

Towards this end, intravitreal sustained-release devices have beeninvestigated. The best known of these devices is the VITRASERT™ganciclovir implant, used in the treatment of cytomegalovirus retinitis.However, implants such as VITRASERT™ require complex and undesirableintraocular surgery, and must be replaced periodically.

Sustained release formulations containing drugs encapsulated inbiodegradable polymer particles are an attractive alternative.Nanoparticle and microparticle formulations can be injected as asuspension, obviating the need for intraocular implantation surgeries.As the polymer particles degrade and/or as the drug diffuses out of thepolymer particles, the drug is released.

Several drawbacks have hampered the successful development of controlledrelease polymeric nanoparticle and microparticle formulations. First, itis often difficult to achieve high and/or controlled drug loading duringparticle formation, particularly for hydrophilic molecules such asdoxorubicin. Grovender T. et al. J. Controlled Release 57(2):171-185(1999). Second, it is difficult to achieve high drug encapsulationefficiency when forming polymeric particle, particularly polymericnanoparticles. Most polymeric particles possess poorly encapsulated drugmolecules on or near the particle surface. As a result, many particlesdisplay an undesirable biphasic drug release pattern. Upon injection,poorly encapsulated drug molecules on or near the surface ofnanoparticles can quickly diffuse into solution, resulting in an initialburst release of drug. In the case of many polymeric nanoparticles, ashigh as 40-80% of the encapsulated drug molecules are released in aburst during the first several or tens of hours followingadministration. After the first 24 to 48 hours, drug release becomessignificantly slower due to the increased diffusion barrier for drugmolecules buried more deeply in polymer particles. Such particles canstill produce a sharp initial increase in drug concentration uponadministration, often to toxic levels.

Therefore, it is an object of the invention to provide polymer-drugconjugates with improved properties for the controlled delivery ofactive agents.

It is also an object of the invention to provide drug formulationscapable of effectively delivering therapeutic levels of one or moreactive agents to the eye for an extended period of time.

It is a further object of the invention to provide improved methods oftreating or preventing diseases or disorders of the eye.

SUMMARY OF THE INVENTION

Non-linear multiblock copolymer-drug conjugates capable of formingnanoparticles, microparticles, and implants with improved properties forcontrolled drug delivery, especially to the eye, are provided. Thepolymer-drug conjugates contain one or more hydrophobic polymer segmentsand one or more hydrophilic polymer segments covalently connectedthrough a multivalent branch point to form a non-linear multiblockcopolymer containing at least three polymeric segments. The polymer-drugconjugates further contain one or more therapeutic, prophylactic, ordiagnostic agents covalently attached to the one or more hydrophobicpolymer segments. By employing a polymer-drug conjugate, particles canbe formed with more controlled drug loading and drug release profiles.In addition, the solubility of the conjugate can be controlled so as tominimize soluble drug concentration and, therefore, toxicity.

The polymer drug conjugates can be represented by the general formulashown below

wherein

A represents, independently for each occurrence, an active agent, withthe proviso that A is not a HIF-1 inhibitor;

X represents, independently for each occurrence, a hydrophobic polymersegment;

Y represents a multivalent branch point;

Z represents, independently for each occurrence, a hydrophilic polymersegment m is an integer between one and twenty and n is an integerbetween zero and twenty.

In some embodiments, m is one or greater and n is zero. In otherembodiments, m and n are one or greater, such that m+n is 2 or greater,such, 4, or 5 or greater.

Preferably, A is a therapeutic or prophylactic agent that is useful forthe treatment or prevention of an ocular disease or disorder, such as ananti-glaucoma agent, anti-angiogenesis agent, anti-infective agent,anti-inflammatory agent, growth factor, immunosuppressant agent, oranti-allergic agent.

The one or more hydrophilic polymer segments can be any hydrophilic,biocompatible, non-toxic polymer or copolymer. In preferred embodiments,the one or more hydrophilic polymer segments contain a poly(alkyleneglycol), such as polyethylene glycol (PEG). In preferred embodiments,the polymer-drug conjugates contain more than one hydrophilic polymersegment.

The one or more hydrophobic polymer segments can be any biocompatible,hydrophobic polymer or copolymer. In preferred embodiments, thehydrophobic polymer or copolymer is biodegradable. In preferredembodiments, the hydrophobic polymer is a polyanhydride, such aspolysebacic anhydride or a copolymer thereof.

The degradation profile of the one or more hydrophobic polymer segmentsmay be selected to influence the release rate of the active agent invivo. For example, the hydrophobic polymer segments can be selected todegrade over a time period from several days to 24 weeks, morepreferably from seven days to eight weeks, preferably from seven days tothree weeks. In other cases, the hydrophobic polymer segments can beselected to degrade over a time period from seven days to 2 years, morepreferably from several days to 56 weeks, more preferably from fourweeks to 56 weeks, most preferably from eight weeks to 28 weeks.

The branch point can be, for example, an organic molecule which containsthree or more functional groups. Preferably, the branch point willcontain at least two different types of functional groups (e.g., one ormore alcohols and one or more carboxylic acids, or one or more halidesand one or more carboxylic acids or one or more amines). In such cases,the different functional groups present on the branch point can beindependently addressed synthetically, permitting the covalentattachment of the hydrophobic and hydrophilic segments to the branchpoint in controlled stoichiometric ratios. In certain embodiments, thebranch point is polycarboxylic acid, such as citric acid, tartaric acid,mucic acid, gluconic acid, or 5-hydroxybenzene-1,2,3,-tricarboxylicacid.

In some embodiments, the branch point connects a single hydrophobicpolymer segment to three hydrophilic polyethylene glycol polymersegments. In certain cases, the polymer-drug conjugate can berepresented by Formula I

wherein

A is an active agent, with the proviso that A is not a HIF-1 inhibitor;

L independently for each occurrence, is absent or an ether (e.g., —O—),thioether (e.g., —S—), secondary amine (e.g., —NH—), tertiary amine(e.g., —NR—), secondary amide (e.g., —NHCO—; —CONH—), tertiary amide(e.g., —NRCO—; —CONR—), secondary carbamate (e.g., —OCONH—; —NHCOO—),tertiary carbamate (e.g., —OCONR—; —NRCOO—), urea (e.g., —NHCONH—;—NRCONH—; —NHCONR—, —NRCONR—), sulfinyl group (e.g., —SO—), or sulfonylgroup (e.g., —SOO—);

R is, individually for each occurrence, an alkyl, cycloalkyl,heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, or heteroarylgroup, optionally substituted with between one and five substituentsindividually selected from alkyl, cyclopropyl, cyclobutyl ether, amine,halogen, hydroxyl, ether, nitrile, CF₃, ester, amide, urea, carbamate,thioether, carboxylic acid, and aryl;

PEG represents a polyethylene glycol chain; and

X represents a hydrophobic polymer segment.

In certain embodiments, the branch point is a citric acid molecule, andthe hydrophilic polymer segments are polyethylene glycol. In such cases,the polymer-drug conjugate can be represented by Formula IA

wherein

A is an active agent, with the proviso that A is not a HIF-1 inhibitor;

D represents, independently for each occurrence, O or NH;

PEG represents a polyethylene glycol chain; and

X represents a hydrophobic polymer segment.

The non-linear multiblock copolymer-drug conjugates form nanoparticles,microparticles, and implants with improved properties for controlleddrug delivery to the eye. Also provided are pharmaceutical compositionscontaining nano- and/or microparticles formed from one or morepolymer-drug conjugates in combination with one or more pharmaceuticallyacceptable excipients, for example, producing a solution or suspensionsuitable for injection or topical application to the eye.

Also provided are methods of administering these pharmaceuticalcompositions, and or an implant containing one or more non-linearmultiblock copolymer-drug conjugates, to treat or prevent a diseases ordisorders of the eye, such as an intraocular neovascular disease (e.g.,wet age-related macular degeneration (AMD), choroidal neovascularization(CNV), and retinal neovascularization (RNV)) or an eye diseaseassociated with inflammation (e.g., uveitis). These formulations andimplants can effectively deliver therapeutic levels of one or moreactive agents to the eye for an extended period of time, in some caseswith decreased side effects when compared to administration of theactive agent alone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B are bar graphs plotting the area of CNV (in mm²) observed inthe eyes of C57BL/6 mice 14 days after rupture of their Bruch's membraneby laser photocoagulation without administration of an anthracycline,and upon administration of varying amounts of doxorubicin ordaunorubicin. In the case of values of CNV measured upon anthracyclineadministration, the area of CNV observed upon anthracycline is plottednext to the area of CNV observed in untreated fellow eyes (FE). The barsrepresent the mean (±SEM) area of choroidal NV. FIG. 1A plots the areaof CNV (in mm²) observed in the eyes of C57BL/6 mice 14 days afterrupture of their Bruch's membrane by laser photocoagulation withoutadministration of an anthracycline (vehicle only injected in both eyesof the mouse (BE), left bar), and upon administration of 10, 1.0, and0.1 μg of daunorubicin (DNR). Eyes injected with 10 μg of DNR showed astatistically significant reduction in the area of CNV (P<0.001, n=10)compared to fellow eyes injected with the vehicle only. Eyes injectedwith 1.0 μg and 0.1 μg of DNR did not show a statistically significantreduction in the area of CNV (for 1.0 μg, P<0.082, n=10; for 0.1 μg,P<0.399, n=10) compared to fellow eyes injected with the vehicle only.FIG. 1B plots the area of CNV (in mm²) observed in the eyes of C57BL/6mice 14 days after rupture of their Bruch's membrane by laserphotocoagulation without administration of an anthracycline (vehicleonly injected in both eyes of the mouse (BE), left bar), and uponadministration of 10, 1.0, and 0.1 μg of doxorubicin (DXR). Eyesinjected with 10 μg of DXR showed a statistically significant reductionin the area of CNV (P<0.001, n=10) compared to fellow eyes injected withthe vehicle only. Eyes injected with 1.0 μg and 0.1 μg of DXR did notshow a statistically significant reduction in the area of CNV (for 1.0μg, P<0.071, n=10; for 0.1 μg, P<0.322, n=10) compared to fellow eyesinjected with the vehicle only. In both FIGS. 2A and 2B, the mean areaof CNV was similar in fellow eyes (FE) and eyes from mice in which botheyes were injected with vehicle only (BE), suggesting that there was nosystemic effect from intraocular injections of the anthracyclines.

FIGS. 2A-B are bar graphs plotting the area of RNV (in mm²) observed inthe eyes of C57BL/6 mice with oxygen-induced ischemic retinopathy fivedays after the administration of a vehicle control (PBS buffer withoutan anthracycline present), and upon administration of varying amounts ofdoxorubicin or daunorubicin. The bars represent the mean (±SEM) area ofRNV. FIG. 2A plots the area of RNV (in mm²) observed in the eyes ofC57BL/6 mice with oxygen-induced ischemic retinopathy five days afterthe administration of a vehicle control (PBS buffer without ananthracycline present, left bar), and upon administration of 1.0, 0.1,and 0.01 μg of daunorubicin (DNR). Eyes injected with 1.0 μg and 0.1 μgof DNR showed a statistically significant reduction in the area of RNV(for 1.0 μg, P<0.001, n=6; for 0.1 μg, P=0.013, n=8). Eyes injected with0.01 μg of DNR did not show a statistically significant reduction in thearea of RNV (P=0.930, n=6). FIG. 2B plots the area of RNV (in mm²)observed in the eyes of C57BL/6 mice with oxygen-induced ischemicretinopathy five days after the administration of a vehicle control (PBSbuffer without an anthracycline present, left bar), and uponadministration of 1.0, 0.1, and 0.01 μg of doxorubicin (DXR). Eyesinjected with 1.0 μg of DXR showed a statistically significant reductionin the area of RNV (P<0.001, n=8). Eyes injected with 0.1 μg and 0.01 μgof DXR did not show a statistically significant reduction in the area ofRNV (for 1.0 μg, P=0.199, n=7; for 0.1 μg, P=0.096, n=8).

FIG. 3A is a graph demonstrating the efficacy of a non-linear multiblockcopolymer-drug conjugate (specifically DXR-PSA-PEG₃ nanoparticles) intreating CNV in a mouse model of CNV. FIG. 3A is a bar graph plottingthe area of CNV (in mm²) observed in the eyes of C57BL/6 mice 14 daysafter rupture of their Bruch's membrane by laser photocoagulationwithout administration of an anthracycline (vehicle only injected inboth eyes of the mouse (BE), left bar), and upon administration of 10,1.0, and 0.1 μg of DXR-PSA-PEG₃ nanoparticles. In the case of values ofCNV measured upon administration of varying amounts of DXR-PSA-PEG₃nanoparticles, the area of CNV observed upon nanoparticle administrationis plotted next to the area of CNV observed in untreated fellow eyes(FE). The bars represent the mean (±SEM) area of CNV. Eyes injected with10 μg, 1.0 μg, and 0.1 μg of DXR-PSA-PEG₃ nanoparticles all showed astatistically significant reduction in the area of CNV (for 10 μg,P<0.001, n=10; for 1.0 μg, P=0.009, n=10; for 0.1 μg, P=0.007, n=10)compared to fellow eyes injected with the vehicle only. One cohort hadthe baseline area of CNV measured, and the remaining mice were treatedby injection of 1 μg of DXR-PSA-PEG₃ nanoparticles in one eye, andinjection of vehicle only in the fellow eye. After an additional sevendays, the area of CNV was measured in the DXR-PSA-PEG₃ andvehicle-treated eyes. FIG. 3B is a bar graph plotting the area of CNV(in mm²) observed seven days after administration of 1 μg ofDXR-PSA-PEG₃ nanoparticles (left bar) and 14 days after laserphotocoagulation rupture of Bruch's membrane. The area of CNV (in mm²)observed seven days after administration of 1 μg of DXR-PSA-PEG₃nanoparticles and 14 days after laser photocoagulation rupture ofBruch's membrane is compared with the area of CNV measured in felloweyes injected with vehicle only (center bar) 14 days after laserphotocoagulation rupture of Bruch's membrane and seven days aftervehicle injection and untreated eyes seven days after laserphotocoagulation rupture of Bruch's membrane (Base line; right bar). Astatistically significant decrease in the area of CNV (P<0.001, n=10)was observed, both relative to fellow eyes injected with vehicle only(center bar) and the base line CNV observed seven days after laserphotocoagulation rupture of Bruch's membrane in untreated eyes (rightbar), demonstrating that DXR-PSA-PEG₃ treatment not only significantlyreduced CNV (compare left and middle bars), but also mediated regressionof existing CNV (compare left and right bars).

FIG. 4 is a graph demonstrating the efficacy of a non-linear multiblockcopolymer-drug conjugate (specifically DXR-PSA-PEG₃ nanoparticles) intreating RNV in mice with oxygen-induced ischemic retinopathy. FIG. 4 isbar graphs plotting the area of RNV (in mm²) observed in the eyes ofC57BL/6 mice with oxygen-induced ischemic retinopathy five days afterthe administration of a vehicle control (PBS buffer without ananthracycline present, right bar), and upon administration of 1 μg ofDXR-PSA-PEG₃ nanoparticles (left bar). The bars represent the mean(±SEM) area of RNV. A statistically significant decrease in the area ofRNV (P<0.001, n=8) was observed relative to fellow eyes injected withvehicle only.

FIGS. 5A-B are bar graphs demonstrating the ability of a non-linearmultiblock copolymer-drug conjugate (specifically DXR-PSA-PEG₃nanoparticles) to suppress subretinal neovascularization (NV) intransgenic mice in which the rhodopsin promoter drives expression ofVEGF in photoreceptors (rho/VEGF mice) for at least 35 days. Atpostnatal day (P) 14, hemizygous rho/VEGF mice were given an intraocularinjection of 10 μg of DXR-PSA-PEG₃ nanoparticles in one eye and vehicleonly (PBS buffer) in the fellow eye. FIG. 5A is a bar graph plotting thearea of NV (in mm²) per retina observed four weeks after intraocularinjection of 10 μg of DXR-PSA-PEG₃ nanoparticles (left bar). Astatistically significant decrease in the area of NV per retina(P=0.042, n=5) was observed relative to fellow eyes injected withvehicle only. FIG. 5B is a bar graph plotting the area of NV (in mm²)per retina observed five weeks after intraocular injection of 10 μg ofDXR-PSA-PEG₃ nanoparticles (left bar). A statistically significantdecrease in the area of NV per retina (P=0.007, n=5) was observedrelative to fellow eyes injected with vehicle only.

FIG. 6A is a graph showing the size distribution by volume ofmicroparticles as determined using a Coulter Multisizer. FIG. 6B is agraph showing the size distribution by volume of nanoparticles asdetermined using a Coulter Multisizer.

FIG. 7A is a graph showing the amount of drug conjugate released (nM)into the aqueous humor (AH) as a function of time (days) frommicroparticles and nanoparticles injected into the eyes of rabbits. FIG.7B is a bar graph that compares the amounts of released DXR drugconjugate (nM) in the aqueous humor (AH) and the vitreous ofparticle-injected rabbit eyes. The time (days) is 105 for thenanoparticle-treated animals and 115 for the microparticle-treatedanimals.

FIG. 8 is a graph showing release of doxorubicin (DXR) conjugate invitro (m) as a function of time (days) for polymer rods containing 10%DXR (●), 30% DXR (▪) and 50% DXR (▴).

FIG. 9A is a graph showing the in vitro release profile of doxorubicin(DXR) conjugate from microparticles prepared from DXR-SA-PEG₃ (▪) andDXR-SA-CPH-PEG (▴). FIG. 9B is a graph showing the in vitro releaseprofile of doxorubicin (DXR) conjugate from microparticles prepared fromDXR-SA-CPH-PEG₃ (▴).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Active Agent”, as used herein, refers to a physiologically orpharmacologically active substance that acts locally and/or systemicallyin the body. An active agent is a substance that is administered to apatient for the treatment (e.g., therapeutic agent), prevention (e.g.,prophylactic agent), or diagnosis (e.g., diagnostic agent) of a diseaseor disorder. “Ophthalmic Drug” or “Ophthalmic Active Agent”, as usedherein, refers to a therapeutic or prophylactic agent that isadministered to a patient to alleviate, delay onset of, or prevent oneor more symptoms of a disease or disorder of the eye, or diagnosticagent useful for imaging or otherwise assessing the eye.

“Effective amount” or “therapeutically effective amount”, as usedherein, refers to an amount of polymer-drug conjugate effective toalleviate, delay onset of, or prevent one or more symptoms of a diseaseor disorder being treated by the active agent, and/or an amount ofpolymer-drug conjugate effective to produce a desired diagnostic signal.In the case of age-related macular degeneration, the effective amount ofthe polymer-drug conjugate delays, reduces, or prevents vision loss in apatient.

“Biocompatible” and “biologically compatible”, as used herein, generallyrefer to materials that are, along with any metabolites or degradationproducts thereof, generally non-toxic to the recipient, and do not causeany significant adverse effects to the recipient. Generally speaking,biocompatible materials are materials which do not elicit a significantinflammatory or immune response when administered to a patient.

“Biodegradable Polymer” as used herein, generally refers to a polymerthat will degrade or erode by enzymatic action or hydrolysis underphysiologic conditions to smaller units or chemical species that arecapable of being metabolized, eliminated, or excreted by the subject.The degradation time is a function of polymer composition, morphology,such as porosity, particle dimensions, and environment.

“Hydrophilic,” as used herein, refers to the property of having affinityfor water. For example, hydrophilic polymers (or hydrophilic polymersegments) are polymers (or polymer segments) which are primarily solublein aqueous solutions and/or have a tendency to absorb water. In general,the more hydrophilic a polymer is, the more that polymer tends todissolve in, mix with, or be wetted by water.

“Hydrophobic,” as used herein, refers to the property of lackingaffinity for, or even repelling water. For example, the more hydrophobica polymer (or polymer segment), the more that polymer (or polymersegment) tends to not dissolve in, not mix with, or not be wetted bywater.

Hydrophilicity and hydrophobicity can be spoken of in relative terms,such as but not limited to a spectrum of hydrophilicity/hydrophobicitywithin a group of polymers or polymer segments. In some embodimentswherein two or more polymers are being discussed, the term “hydrophobicpolymer” can be defined based on the polymer's relative hydrophobicitywhen compared to another, more hydrophilic polymer.

“Nanoparticle”, as used herein, generally refers to a particle having adiameter, such as an average diameter, from about 10 nm up to but notincluding about 1 micron, preferably from 100 nm to about 1 micron. Theparticles can have any shape. Nanoparticles having a spherical shape aregenerally referred to as “nanospheres”.

“Microparticle”, as used herein, generally refers to a particle having adiameter, such as an average diameter, from about 1 micron to about 100microns, preferably from about 1 to about 50 microns, more preferablyfrom about 1 to about 30 microns, most preferably from about 1 micron toabout 10 microns. The microparticles can have any shape. Microparticleshaving a spherical shape are generally referred to as “microspheres”.

“Molecular weight” as used herein, generally refers to the relativeaverage chain length of the bulk polymer, unless otherwise specified. Inpractice, molecular weight can be estimated or characterized usingvarious methods including gel permeation chromatography (GPC) orcapillary viscometry. GPC molecular weights are reported as theweight-average molecular weight (Mw) as opposed to the number-averagemolecular weight (Mn). Capillary viscometry provides estimates ofmolecular weight as the inherent viscosity determined from a dilutepolymer solution using a particular set of concentration, temperature,and solvent conditions.

“Mean particle size” as used herein, generally refers to the statisticalmean particle size (diameter) of the particles in a population ofparticles. The diameter of an essentially spherical particle may referto the physical or hydrodynamic diameter. The diameter of anon-spherical particle may refer preferentially to the hydrodynamicdiameter. As used herein, the diameter of a non-spherical particle mayrefer to the largest linear distance between two points on the surfaceof the particle. Mean particle size can be measured using methods knownin the art, such as dynamic light scattering.

“Monodisperse” and “homogeneous size distribution”, are usedinterchangeably herein and describe a population of nanoparticles ormicroparticles where all of the particles are the same or nearly thesame size. As used herein, a monodisperse distribution refers toparticle distributions in which 90% or more of the distribution lieswithin 15% of the median particle size, more preferably within 10% ofthe median particle size, most preferably within 5% of the medianparticle size.

“Pharmaceutically Acceptable”, as used herein, refers to compounds,carriers, excipients, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

“Branch point”, as used herein, refers to a portion of a polymer-drugconjugate that serves to connect one or more hydrophilic polymersegments to one or more hydrophobic polymer segments.

“Implant,” as generally used herein, refers to a polymeric device orelement that is structured, sized, or otherwise configured to beimplanted, preferably by injection or surgical implantation, in aspecific region of the body so as to provide therapeutic benefit byreleasing one or more HIF-1 inhibitors over an extended period of timeat the site of implantation. For example, intraocular implants arepolymeric devices or elements that are structured, sized, or otherwiseconfigured to be placed in the eye, preferably by injection or surgicalimplantation, and to treat one or more diseases or disorders of the eyeby releasing one or more HIF-1 inhibitors over an extended period.Intraocular implants are generally biocompatible with physiologicalconditions of an eye and do not cause adverse side effects. Generally,intraocular implants may be placed in an eye without disrupting visionof the eye.

Ranges of values defined herein include all values within the range aswell as all sub-ranges within the range. For example, if the range isdefined as an integer from 0 to 10, the range encompasses all integerswithin the range and any and all subranges within the range, e.g., 1-10,1-6, 2-8, 3-7, 3-9, etc.

II. Non-Linear Multiblock Copolymer-Drug Conjugates

Non-linear multiblock copolymer-drug conjugates can be used to formnanoparticles, microparticles, and implants (e.g., rods, discs, wafers,etc.) useful for the delivery to the eye. The polymer-drug conjugatescontain one or more hydrophobic polymer segments and one or morehydrophilic polymer segments covalently connected through a multivalentbranch point to form a non-linear multiblock copolymer containing atleast three polymeric segments. The polymer-drug conjugates furthercontain one or more therapeutic, prophylactic, or diagnostic agentscovalently attached to the one or more hydrophobic polymer segments. Byemploying a polymer-drug conjugate, particles can be formed with morecontrolled drug loading and drug release profiles. In addition, thesolubility of the conjugate can be controlled so as to minimize solubledrug concentration and, therefore, toxicity.

A. Structure of the Non-Linear Multiblock Copolymer-Drug Conjugates

Non-linear multiblock copolymer-drug conjugates are provided whichcontain an active agent covalently attached to one or more hydrophobicpolymer segments which are covalently attached to one or morehydrophilic polymer segments.

The polymer drug conjugates can be represented by the general formulashown below

wherein

A represents, independently for each occurrence, an active agent, withthe proviso that A is not a HIF-1 inhibitor;

X represents, independently for each occurrence, a hydrophobic polymersegment;

Y represents a multivalent branch point;

Z represents, independently for each occurrence, a hydrophilic polymersegment m is an integer between one and twenty and n is an integerbetween zero and twenty.

In some embodiments, m is one or greater and n is zero. In otherembodiments, m and n are one or greater, such that m+n is 2 or greater,such, 4, or 5 or greater.

In some embodiments, the conjugate is a mixture of the conjugates above,where, for some conjugates, n is an integer value other than 0 and forother conjugate, n=0.

A can be, independently for each occurrence, an active agent which isuseful for the treatment, diagnosis, or prophylaxis of a disease ordisorder of the eye (jointly referred to herein as “drug”), with theproviso that A is not a HIF-1 inhibitor.

The one or more hydrophobic polymer segments, independently, can be anybiocompatible hydrophobic polymer or copolymer. In some cases, the oneor more hydrophobic polymer segments are also biodegradable. Examples ofsuitable hydrophobic polymers include polyesters such as polylacticacid, polyglycolic acid, or polycaprolactone, polyanhydrides, such aspolysebacic anhydride, and copolymers thereof. In certain embodiments,the hydrophobic polymer is a polyanhydride, such as polysebacicanhydride or a copolymer thereof.

The one or more hydrophilic polymer segments can be any hydrophilic,biocompatible, non-toxic polymer or copolymer. The hydrophilic polymersegment can be, for example, a poly(alkylene glycol), a polysaccharide,poly(vinyl alcohol), polypyrrolidone, a polyoxyethylene block copolymer(PLURONIC®) or a copolymers thereof. In preferred embodiments, the oneor more hydrophilic polymer segments are, or are composed of,polyethylene glycol (PEG).

In some cases, the polymer-drug conjugate contains only one hydrophilicpolymer segment. In preferred embodiments, the polymer-drug conjugatecontains more than one hydrophilic polymer chain. In certainembodiments, the polymer-drug conjugate contains between two and six,more preferably between three and five, hydrophilic polymer chains. Inone embodiment, the polymer drug conjugate contains three hydrophilicpolymer segments.

Preferably, the combined molecular weight of the one or more hydrophilicpolymer segments will be larger than the molecular weight of the one ormore hydrophobic polymer segments. In some cases, the combined molecularweight of the one or more hydrophilic polymer segments is at least threetimes, more preferably at least five times, most preferably at least tentimes greater than the molecular weight of the one or more hydrophobicpolymer segment.

The branch point can be, for example, an organic molecule which containsthree or more functional groups. Preferably, the branch point willcontain at least two different types of functional groups (e.g., one ormore alcohols and one or more carboxylic acids, or one or more halidesand one or more carboxylic acids). In such cases, the differentfunctional groups present on the branch point can be independentlyaddressed synthetically, permitting the covalent attachment of thehydrophobic and hydrophilic segments to the branch point in controlledstoichiometric ratios. In certain embodiments, the branch point ispolycarboxylic acid, such as citric acid, tartaric acid, mucic acid,gluconic acid, or 5-hydroxybenzene-1,2,3,-tricarboxylic acid.

In certain embodiments, the polymer-drug conjugate is formed from asingle hydrophobic polymer segment and two or more hydrophilic polymersegments covalently connected via a multivalent branch point. Exemplarypolymer-drug conjugates of this type are represented by the generalformula shown belowA-X—Y

Z)_(n)wherein

A represents an active agent, with the proviso that A is not a HIF-1inhibitor;

X represents a hydrophobic polymer segment;

Y represents a branch point;

Z represents, independently for each occurrence, a hydrophilic polymersegment; and

n is an integer between one or two and 300, more preferably between oneor two and fifty, more preferably between one or two and thirty, mostpreferably between one or two and ten.

In certain embodiments, the polymer-drug conjugate contains between twoand six, more preferably between three and five hydrophilic polymerchains. In one embodiment, the polymer drug conjugate contains threehydrophilic polymer segments.

The branch point can be, for example, an organic molecule which containsmultiple functional groups. Preferably, the branch point will contain atleast two different types of functional groups (e.g., an alcohol andmultiple carboxylic acids, or a carboxylic acid and multiple alcohols).In certain embodiments, the branch point is polycarboxylic acid, such asa citric acid molecule.

In some embodiments, the branch point connects a single hydrophobicpolymer segment to three hydrophilic polyethylene glycol polymersegments. In certain cases, the polymer-drug conjugate can berepresented by Formula I

wherein

A is an active agent, with the proviso that A is not a HIF-1 inhibitor;

L independently for each occurrence, is absent or an ether (e.g., —O—),thioether (e.g., —S—), secondary amine (e.g., —NH—), tertiary amine(e.g., —NR—), secondary amide (e.g., —NHCO—; —CONH—), tertiary amide(e.g., —NRCO—; —CONR—), secondary carbamate (e.g., —OCONH—; —NHCOO—),tertiary carbamate (e.g., —OCONR—; —NRCOO—), urea (e.g., —NHCONH—;—NRCONH—; —NHCONR—, —NRCONR—), sulfinyl group (e.g., —SO—), or sulfonylgroup (e.g., —SOO—);

R is, individually for each occurrence, an alkyl, cycloalkyl,heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, or heteroarylgroup, optionally substituted with between one and five substituentsindividually selected from alkyl, cyclopropyl, cyclobutyl ether, amine,halogen, hydroxyl, ether, nitrile, CF₃, ester, amide, urea, carbamate,thioether, carboxylic acid, and aryl;

PEG represents a polyethylene glycol chain; and

X represents a hydrophobic polymer segment.

In certain embodiments, the branch point is a citric acid molecule, andthe hydrophilic polymer segments are polyethylene glycol. In such cases,the polymer-drug conjugate can be represented by Formula IA

wherein

A is an active agent, with the proviso that A is not a HIF-1 inhibitor;

D represents, independently for each occurrence, O or NH;

PEG represents a polyethylene glycol chain; and

X represents a hydrophobic polymer segment.

In some embodiments, the polymer drug conjugate is defined by thefollowing formulaA-Xwherein

A is an active agent, with the proviso that A is not a HIF-1 inhibitor;and

X is a hydrophobic polymer segment, preferably a polyanhydride.

B. Active Agents

Non-linear multiblock copolymer-drug conjugates contain a therapeutic,diagnostic, and/or prophylactic agent. The active agent can be a smallmolecule active agent and/or a biomolecule, such as an enzyme, protein,growth factor, polypeptide, polysaccharide, lipid, or nucleic acid.Suitable small molecule active agents include organic and organometalliccompounds. In some instances, the small molecule active agent has amolecular weight of less than about 2000 g/mol, preferably less thanabout 1500 g/mol, more preferably less than about 1200 g/mol, mostpreferably less than about 1000 g/mol. In other embodiments, the smallmolecule active agent has a molecular weight less than about 500 g/mol.The small molecule active agent can be a hydrophilic, hydrophobic, oramphiphilic compound. Biomolecules typically have a molecular weight ofgreater than about 2000 g/mol and may be composed of repeat units suchas amino acids (peptide, proteins, enzymes, etc.) or nitrogenous baseunits (nucleic acids).

In preferred embodiments, the active agent is an ophthalmic drug. Inparticular embodiments, the active agent is a drug used to treat,prevent or diagnose a disease or disorder of the posterior segment eye.Non-limiting examples of ophthalmic drugs include anti-glaucoma agents,anti-angiogenesis agents, anti-infective agents, anti-inflammatoryagents, growth factors, immunosuppressant agents, anti-allergic agents,and combinations thereof.

Representative anti-glaucoma agents include prostaglandin analogs (suchas travoprost, bimatoprost, and latanoprost), beta-andrenergic receptorantagonists (such as timolol, betaxolol, levobetaxolol, and carteolol),alpha-2 adrenergic receptor agonists (such as brimonidine andapraclonidine), carbonic anhydrase inhibitors (such as brinzolamide,acetazolamine, and dorzolamide), miotics (i.e., parasympathomimetics,such as pilocarpine and ecothiopate), seretonergics muscarinics,dopaminergic agonists, and adrenergic agonists (such as apraclonidineand brimonidine).

Representative anti-angiogenesis agents include, but are not limited to,antibodies to vascular endothelial growth factor (VEGF) such asbevacizumab (AVASTIN®) and rhuFAb V2 (ranibizumab, LUCENTIS®), and otheranti-VEGF compounds; MACUGEN® (pegaptanim sodium, anti-VEGF aptamer orEYE001) (Eyetech Pharmaceuticals); pigment epithelium derived factor(s)(PEDF); COX-2 inhibitors such as celecoxib (CELEBREX®) and rofecoxib(VIOXX®); interferon alpha; interleukin-12 (IL-12); thalidomide(THALOMID®) and derivatives thereof such as lenalidomide (REVLIMID®);squalamine; endostatin; angiostatin; ribozyme inhibitors such asANGIOZYME® (Sirna Therapeutics); multifunctional antiangiogenic agentssuch as NEOVASTAT® (AE-941) (Aeterna Laboratories, Quebec City, Canada);receptor tyrosine kinase (RTK) inhibitors such as sunitinib (SUTENT®);tyrosine kinase inhibitors such as sorafenib (Nexavar®) and erlotinib(Tarceva®); antibodies to the epidermal grown factor receptor such aspanitumumab (VECTIBIX®) and cetuximab (ERBITUX®), as well as otheranti-angiogenesis agents known in the art.

Anti-infective agents include antiviral agents, antibacterial agents,antiparasitic agents, and anti-fungal agents. Representative antiviralagents include ganciclovir and acyclovir. Representative antibioticagents include aminoglycosides such as streptomycin, amikacin,gentamicin, and tobramycin, ansamycins such as geldanamycin andherbimycin, carbacephems, carbapenems, cephalosporins, glycopeptidessuch as vancomycin, teicoplanin, and telavancin, lincosamides,lipopeptides such as daptomycin, macrolides such as azithromycin,clarithromycin, dirithromycin, and erythromycin, monobactams,nitrofurans, penicillins, polypeptides such as bacitracin, colistin andpolymyxin B, quinolones, sulfonamides, and tetracyclines.

In some cases, the active agent is an anti-allergic agent such asolopatadine and epinastine.

Anti-inflammatory agents include both non-steroidal and steroidalanti-inflammatory agents. Suitable steroidal active agents includeglucocorticoids, progestins, mineralocorticoids, and corticosteroids.

The ophthalmic drug may be present in its neutral form, or in the formof a pharmaceutically acceptable salt. In some cases, it may bedesirable to prepare a formulation containing a salt of an active agentdue to one or more of the salt's advantageous physical properties, suchas enhanced stability or a desirable solubility or dissolution profile.

Generally, pharmaceutically acceptable salts can be prepared by reactionof the free acid or base forms of an active agent with a stoichiometricamount of the appropriate base or acid in water or in an organicsolvent, or in a mixture of the two; generally, non-aqueous media likeether, ethyl acetate, ethanol, isopropanol, or acetonitrile arepreferred. Pharmaceutically acceptable salts include salts of an activeagent derived from inorganic acids, organic acids, alkali metal salts,and alkaline earth metal salts as well as salts formed by reaction ofthe drug with a suitable organic ligand (e.g., quaternary ammoniumsalts). Lists of suitable salts are found, for example, in Remington'sPharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins,Baltimore, Md., 2000, p. 704. Examples of ophthalmic drugs sometimesadministered in the form of a pharmaceutically acceptable salt includetimolol maleate, brimonidine tartrate, and sodium diclofenac.

In some cases, the active agent is a diagnostic agent imaging orotherwise assessing the eye. Exemplary diagnostic agents includeparamagnetic molecules, fluorescent compounds, magnetic molecules, andradionuclides, x-ray imaging agents, and contrast media.

C. Hydrophobic Polymer Segment

The non-linear multiblock copolymer-drug conjugates described herein cancontain one or more hydrophobic polymer segments. The one or morehydrophobic polymer segments can be homopolymers or copolymers.Preferably, the one or more hydrophobic polymer segments arebiodegradable.

Examples of suitable hydrophobic polymers include polyhydroxyacids suchas poly(lactic acid), poly(glycolic acid), and poly(lacticacid-co-glycolic acids); polyhydroxyalkanoates such aspoly-3-hydroxybutyrate or poly-4-hydroxybutyrate; polycaprolactones;poly(orthoesters); polyanhydrides; poly(phosphazenes);poly(hydroxyalkanoates); poly(lactide-co-caprolactones); polycarbonatessuch as tyrosine polycarbonates; polyamides (including synthetic andnatural polyamides), polypeptides, and poly(amino acids);polyesteramides; polyesters; poly(dioxanones); poly(alkylene alkylates);hydrophobic polyethers; polyurethanes; polyetheresters; polyacetals;polycyanoacrylates; polyacrylates; polymethylmethacrylates;polysiloxanes; poly(oxyethylene)/poly(oxypropylene) copolymers;polyketals; polyphosphates; polyhydroxyvalerates; polyalkylene oxalates;polyalkylene succinates; poly(maleic acids), as well as copolymersthereof.

In preferred embodiments, the one or more hydrophobic polymer segmentsare polyanhydrides or copolymers thereof. The polyanhydrides can bealiphatic polyanhydrides, unsaturated polyanhydrides, or aromaticpolyanhydrides. Representative polyanhydrides include polyadipicanhydride, polyfumaric anhydride, polysebacic anhydride, polymaleicanhydride, polymalic anhydride, polyphthalic anhydride, polyisophthalicanhydride, polyaspartic anhydride, polyterephthalic anhydride,polyisophthalic anhydride, poly carboxyphenoxypropane anhydride,polycarboxyphenoxyhexane anhydride, as well as copolymers of thesepolyanhydrides with other polyanhydrides at different mole ratios. Othersuitable polyanhydrides are disclosed in U.S. Pat. Nos. 4,757,128,4,857,311, 4,888,176, and 4,789,724. The one or more hydrophobic polymersegments can also be polyanhydride copolymers, such as apoly(ester-anhydrides) or poly(amide-anhydrides). See, for example, U.S.Pat. No. 5,756,652 and U.S. Patent Application No. US 2010/0260703.

In certain embodiments, the hydrophobic polymer segment is polysebacicanhydride. In certain embodiments, the hydrophobic polymer segment ispoly(1,6-bis(p-carboxyphenoxy)hexane-co-sebacic acid) (poly(CPH-SA). Incertain embodiments, the hydrophobic polymer segment ispoly(1,3-bis(p-carboxyphenoxy)propane-co-sebacic acid) (poly(CPP-SA).

In preferred embodiments, the one or more hydrophobic polymer segmentsare biodegradable. In cases where the one or more hydrophobic polymersegments are biodegradable, the polymer degradation profile may beselected to influence the release rate of the active agent in vivo. Forexample, the one or more hydrophobic polymer segments can be selected todegrade over a time period from seven days to 24 weeks, more preferablyfrom seven days to eight weeks, preferably from seven days to threeweeks. In other cases, the hydrophobic polymer segments can be selectedto degrade over a time period from seven days to 2 years, morepreferably from seven days to 56 weeks, more preferably from four weeksto 56 weeks, most preferably from eight weeks to 28 weeks.

The molecular weight of the one or more hydrophobic polymer segments canbe varied to prepare polymer-drug conjugates that form particles havingproperties, such as drug release rate, optimal for specificapplications. The one or more hydrophobic polymer segments can have amolecular weight of about 150 Da to 1 MDa. In certain embodiments, thehydrophobic polymer segment has a molecular weight of between about 1kDa and about 100 kDa, more preferably between about lkDa and about 50kDa, most preferably between about 1 kDa and about 25 kDa.

In some cases, the one or more hydrophobic polymer segments have acombined average molecular weight that is less than the combined averagemolecular weight of the one or more hydrophilic polymer segments of thepolymer-drug conjugate. In a preferred embodiment, the one or morehydrophobic polymer segments each have an average molecular weight ofless than about 5 kDa.

D. Hydrophilic Polymers

The non-linear multiblock copolymer-drug conjugates described hereinalso contain one or more hydrophilic polymer segments. Preferably, thepolymer-drug conjugates contain more than one hydrophilic polymersegment. In some embodiments, the polymer-drug conjugate containsbetween two and six, more preferably between three and five, hydrophilicpolymer segments. In certain embodiments, the polymer drug conjugatecontains three hydrophilic polymer segment.

Each hydrophilic polymer segment can independently contain anyhydrophilic, biocompatible (i.e., it does not induce a significantinflammatory or immune response), non-toxic polymer or copolymer.Examples of suitable polymers include, but are not limited to,poly(alkylene glycols) such as polyethylene glycol (PEG), poly(propyleneglycol) (PPG), and copolymers of ethylene glycol and propylene glycol,poly(oxyethylated polyol), poly(olefinic alcohol),polyvinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(amino acids),poly(hydroxy acids), poly(vinyl alcohol), and copolymers, terpolymers,and mixtures thereof.

In preferred embodiments, the one or more hydrophilic polymer segmentscontain a poly(alkylene glycol) chain. The poly(alkylene glycol) chainsmay contain between 1 and 500 repeat units, more preferably between 40and 500 repeat units. Suitable poly(alkylene glycols) includepolyethylene glycol, polypropylene 1,2-glycol, poly(propylene oxide),polypropylene 1,3-glycol, and copolymers thereof.

In some embodiments, the one or more hydrophilic polymer segments arecopolymers containing one or more blocks of polyethylene oxide (PEO)along with one or more blocks composed of other biocompatible polymers(for example, poly(lactide), poly(glycolide),poly(lactide-co-glycolide), or polycaprolactone). The one or morehydrophilic polymer segments can be copolymers containing one or moreblocks of PEO along with one or more blocks containing polypropyleneoxide (PPO). Specific examples include triblock copolymers ofPEO—PPO-PEO, such as POLOXAMERS™ and PLURONICS™.

In preferred embodiments, the one or more hydrophilic polymer segmentsare PEG chains. In such cases, the PEG chains can be linear or branched,such as those described in U.S. Pat. No. 5,932,462. In certainembodiments, the PEG chains are linear.

Each of the one or more hydrophilic polymer segments can independentlyhave a molecular weight of about 300 Da to 1 MDa. The hydrophilicpolymer segment may have a molecular weight ranging between any of themolecular weights listed above. In certain embodiments, each of the oneor more hydrophilic polymer segments has a molecular weight of betweenabout 1 kDa and about 20 kDa, more preferably between about 1 kDa andabout 15 kDa, most preferably between about lkDa and about 10 kDa. In apreferred embodiment, each of the one or more hydrophilic polymersegments has a molecular weight of about 5 kDa.

E. Branch Points

The non-linear multiblock copolymer-drug conjugates described hereincontain a branch point which connects the one or more hydrophilicpolymer segments and the one or more hydrophobic polymer segments. Thebranch point can be any organic, inorganic, or organometallic moietywhich is polyvalent, so as to provide more than two points ofattachment. In preferred embodiments, the branch point is an organicmolecule which contains multiple functional groups.

The functional groups may be any atom or group of atoms that contains atleast one atom that is neither carbon nor hydrogen, with the provisothat the groups must be capable of reacting with the hydrophobic andhydrophilic polymer segments. Suitable functional groups includehalogens (bromine, chlorine, and iodine); oxygen-containing functionalgroups such as a hydroxyls, epoxides, carbonyls, aldehydes, ester,carboxyls, and acid chlorides; nitrogen-containing functional groupssuch as amines and azides; and sulfur-containing groups such as thiols.The functional group may also be a hydrocarbon moiety which contains oneor more non-aromatic pi-bonds, such as an alkyne, alkene, or diene.Preferably, the branch point will contain at least two different typesof functional groups (e.g., one or more alcohols and one or morecarboxylic acids, or one or more halides and one or more alcohols). Insuch cases, the different functional groups present on the branch pointcan be independently addressed synthetically, permitting the covalentattachment of the hydrophobic and hydrophilic segments to the branchpoint in controlled stoichiometric ratios.

Following reaction of the hydrophobic and hydrophilic polymer segmentswith functional groups on the branch point, the one or more hydrophobicpolymer segments and the one or more hydrophilic polymer segments willbe covalently joined to the branch point via linking moieties. Theidentity of the linking moieties will be determined by the identity ofthe functional group and the reactive locus of the hydrophobic andhydrophilic polymer segments (as these elements react to form thelinking moiety or a precursor of the linking moiety). Examples ofsuitable linking moieties that connect a the polymer segments to thebranch point include secondary amides (—CONH—), tertiary amides(—CONR—), secondary carbamates (—OCONH—; —NHCOO—), tertiary carbamates(—OCONR—; —NRCOO—), ureas (—NHCONH—; —NRCONH—; —NHCONR—, —NRCONR—),carbinols (—CHOH—, —CROH—), ethers (—O—), and esters (—COO—, —CH₂O₂C—,CHRO₂C—), wherein R is an alkyl group, an aryl group, or a heterocyclicgroup. In certain embodiments, the polymer segments are connected to thebranch point via an ester (—COO—, —CH₂O₂C—, CHRO₂C—), a secondary amide(—CONH—), or a tertiary amide (—CONR—), wherein R is an alkyl group, anaryl group, or a heterocyclic group.

In certain embodiments, the branch point is polycarboxylic acid, such ascitric acid, tartaric acid, mucic acid, gluconic acid, or5-hydroxybenzene-1,2,3,-tricarboxylic acid. Exemplary branch pointsinclude the following organic compounds:

F. Synthesis of Non-Linear Multiblock Copolymer-Drug Conjugates

Non-linear multiblock copolymer-drug conjugates can be prepared usingsynthetic methods known in the art. Representative methodologies for thepreparation of polymer-drug conjugates are discussed below. Theappropriate route for synthesis of a given polymer-drug conjugate can bedetermined in view of a number of factors, such as the structure of thepolymer-drug conjugate, the identity of the polymers which make up theconjugate, the identity of the active agent, as well as the structure ofthe compound as a whole as it relates to compatibility of functionalgroups, protecting group strategies, and the presence of labile bonds.

In addition to the synthetic methodologies discussed below, alternativereactions and strategies useful for the preparation of the polymer-dugconjugates disclosed herein are known in the art. See, for example,March, “Advanced Organic Chemistry,” 5^(th) Edition, 2001,Wiley-Interscience Publication, New York).

Generally, non-linear multiblock copolymer-drug conjugates are preparedby first sequentially attaching the one or more hydrophobic polymersegments and the one or more hydrophilic polymer segments to a branchpoint to form the polymeric portion of the polymer-drug conjugate.Following assembly of the polymeric components of the polymer-drugconjugate, one or more active agents can then be covalently attached tothe one or more hydrophobic polymer segments.

For example, Schemes 1 and 2 illustrate the synthesis of a polymer-drugconjugate (DXR-PSA-PEG₃) containing a citric acid branch pointfunctionalized with three hydrophilic PEG segments and a singlehydrophobic poly(sebacic anhydride) polymer segment. Doxorubicin (DXR)is bound to the hydrophobic polymer segment.

As shown in scheme 1, citric acid is first reacted with CH₃O-PEG-NH₂ inthe presence of N,N′-dicyclohexylcarbodiimide (DCC) and a catalyticamount of 4-dimethylaminopyridine (DMAP), forming amide linkages betweenthe PEG chains and the three carboxylic acid residues of the citric acidbranch point. The resulting compound is then reacted with an acylatedpolysebacic acid precursor (PreSA), and polymerized under anhydroushot-melt polymerization conditions. As shown in Scheme 2, the resultingpolymer (PSA-PEG₃) is then reacted with doxorubicin to form thepolymer-drug conjugate (DXR-PSA-PEG₃).

III. Particles and Implants Formed from Polymer-Drug Conjugates

Non-linear multiblock copolymer-drug conjugates can be formed intomicroparticles, nanoparticles, and implants using a variety oftechniques known in the art. An appropriate method for particle orimplant formation can be selected in view of the physical and chemicalproperties of the one or more polymer-drug conjugates used to form theparticles/implants (i.e., stability and solubility) as well as thedesired particle/implant size and morphology.

A. Particle Morphology

Microparticles and nanoparticles can be formed from one or morepolymer-drug conjugates. In some cases, particles are formed from asingle polymer-drug conjugate (i.e., the particles are formed from apolymer-drug conjugate which contains the same active agent, hydrophobicpolymer segment or segments, branch point (when present), andhydrophilic polymer segment or segments).

In other embodiments, the particles are formed from a mixture of two ormore different polymer-drug conjugates. For example, particles may beformed from two or more polymer-drug conjugates containing differentactive agents and the same hydrophobic polymer segment or segments,branch point (when present), and hydrophilic polymer segment orsegments. Such particles can be used, for example, to co-administer twoor more active agents. In other cases, the particles are formed from twoor more polymer-drug conjugates containing the same active agent, anddifferent hydrophobic polymer segments, branch points (when present),and/or hydrophilic polymer segments. Such particles can be used, forexample, to vary the release rate of active agents over time. Theparticles can also be formed from two or more polymer-drug conjugatescontaining different active agents and different hydrophobic polymersegments, branch points (when present), and/or hydrophilic polymersegments.

Particles can also be formed from blends of polymer-drug conjugates withone or more additional polymers. In these cases, the one or moreadditional polymers can be any of the non-biodegradable or biodegradablepolymers described in Section C below, although biodegradable polymersare preferred. In these embodiments, the identity and quantity of theone or more additional polymers can be selected, for example, toinfluence particle stability, i.e. that time required for distributionto the site where delivery is desired, and the time desired fordelivery.

Particles having an average particle size of between 10 nm and 1000microns are useful in the compositions described herein. In preferredembodiments, the particles have an average particle size of between 10nm and 100 microns, more preferably between about 100 nm and about 50microns, more preferably between about 200 nm and about 50 microns. Incertain embodiments, the particles are nanoparticles having a diameterof between 500 and 700 nm. The particles can have any shape but aregenerally spherical in shape.

In some embodiments, the population of particles formed from one or morepolymer-drug conjugates is a monodisperse population of particles. Inother embodiments, the population of particles formed from one or morepolymer-drug conjugates is a polydisperse population of particles. Insome instances where the population of particles formed from one or morepolymer-drug conjugates is polydisperse population of particles, greaterthat 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the particle sizedistribution lies within 10% of the median particle size.

Preferably, particles formed from one or more polymer-drug conjugatescontain significant amounts of a hydrophilic polymer, such as PEG, ontheir surface.

B. Methods of Forming Microparticles and Nanoparticles

Microparticle and nanoparticles formed from polymer-drug conjugates canbe prepared using any suitable method for the formation of polymermicro- or nanoparticles known in the art. The method employed forparticle formation will depend on a variety of factors, including thecharacteristics of the polymers present in the polymer-drug conjugateand the desired particle size and size distribution. The type of activeagent(s) present in the particle-drug conjugate may also be a factor assome agents are unstable in the presence of certain solvents, in certaintemperature ranges, and/or in certain pH ranges.

In circumstances where a monodisperse population of particles isdesired, the particles may be formed using a method which produces amonodisperse population of nanoparticles. Alternatively, methodsproducing polydisperse nanoparticle distributions can be used, and theparticles can beseparated following particle formation to provide apopulation of particles having the desired average particle size andparticle size distribution. Such separations can be performed usingmethods known in the art, such as sieving.

Common techniques for preparing microparticles and nanoparticlesinclude, but are not limited to, solvent evaporation, hot melt particleformation, solvent removal, spray drying, phase inversion, coacervation,and low temperature casting. Suitable methods of particle formulationare briefly described below. Pharmaceutically acceptable excipients,including pH modifying agents, disintegrants, preservatives, andantioxidants, can optionally be incorporated into the particles duringparticle formation.

1. Solvent Evaporation

In this method, the polymer-drug conjugate is dissolved in a volatileorganic solvent, such as methylene chloride. The organic solutioncontaining the polymer-drug conjugate is then suspended in an aqueoussolution that contains a surface active agent such as poly(vinylalcohol). The resulting emulsion is stirred until most of the organicsolvent evaporated, leaving solid nanoparticles. The resultingnanoparticles are washed with water and dried overnight in alyophilizer. Nanoparticles with different sizes and morphologies can beobtained by this method.

Polymer-drug conjugates which contain labile polymers, such as certainpolyanhydrides, may degrade during the fabrication process due to thepresence of water. For these polymers, the following two methods, whichare performed in completely anhydrous organic solvents, can be used.

2. Hot Melt Particle Formation

In this method, the polymer-drug conjugate is first melted, and thensuspended in a non-miscible solvent (like silicon oil), and, withcontinuous stirring, heated to 5° C. above the melting point of thepolymer-drug conjugate. Once the emulsion is stabilized, it is cooleduntil the polymer-drug conjugate particles solidify. The resultingnanoparticles are washed by decantation with a suitable solvent, such aspetroleum ether, to give a free-flowing powder. The external surfaces ofparticles prepared with this technique are usually smooth and dense. Hotmelt particle formation can be used to prepare particles containingpolymer-drug conjugates which are hydrolytically unstable, such ascertain polyanhydrides. Preferably, the polymer-drug conjugate used toprepare microparticles via this method will have an overall molecularweight of less than 75,000 Daltons.

3. Solvent Removal

Solvent removal can also be used to prepare particles from polymer-drugconjugates that are hydrolytically unstable. In this method, thepolymer-drug conjugate is dispersed or dissolved in a volatile organicsolvent such as methylene chloride. This mixture is then suspended bystirring in an organic oil (such as silicon oil) to form an emulsion.Solid particles form from the emulsion, which can subsequently beisolated from the supernatant. The external morphology of spheresproduced with this technique is highly dependent on the identity of thepolymer-drug conjugate.

4. Spray Drying

In this method, the polymer-drug conjugate is dissolved in an organicsolvent such as methylene chloride. The solution is pumped through amicronizing nozzle driven by a flow of compressed gas, and the resultingaerosol is suspended in a heated cyclone of air, allowing the solvent toevaporate from the microdroplets, forming particles. Particles rangingbetween 0.1-10 microns can be obtained using this method.

5. Phase Inversion

Particles can be formed from polymer-drug conjugates using a phaseinversion method. In this method, the polymer-drug conjugate isdissolved in a “good” solvent, and the solution is poured into a strongnon solvent for the polymer-drug conjugate to spontaneously produce,under favorable conditions, microparticles or nanoparticles. The methodcan be used to produce nanoparticles in a wide range of sizes,including, for example, about 100 nanometers to about 10 microns,typically possessing a narrow particle size distribution.

6. Coacervation

Techniques for particle formation using coacervation are known in theart, for example, in GB-B-929 406; GB-B-929 40 1; and U.S. Pat. Nos.3,266,987, 4,794,000, and 4,460,563. Coacervation involves theseparation of a polymer-drug conjugate solution into two immiscibleliquid phases. One phase is a dense coacervate phase, which contains ahigh concentration of the polymer-drug conjugate, while the second phasecontains a low concentration of the polymer-drug conjugate. Within thedense coacervate phase, the polymer-drug conjugate forms nanoscale ormicroscale droplets, which harden into particles. Coacervation may beinduced by a temperature change, addition of a non-solvent or additionof a micro-salt (simple coacervation), or by the addition of anotherpolymer thereby forming an interpolymer complex (complex coacervation).

7. Low Temperature Casting

Methods for very low temperature casting of controlled releasemicrospheres are described in U.S. Pat. No. 5,019,400 to Gombotz et al.In this method, the polymer-drug conjugate is dissolved in a solvent.The mixture is then atomized into a vessel containing a liquidnon-solvent at a temperature below the freezing point of thepolymer-drug conjugate solution which freezes the polymer-drug conjugatedroplets. As the droplets and non-solvent for the polymer-drug conjugateare warmed, the solvent in the droplets thaws and is extracted into thenon-solvent, hardening the microspheres.

C. Implants Formed from Polymer-Drug Conjugates

Implants can be formed from one or more non-linear multiblockcopolymer-drug conjugates. In preferred embodiments, the implants areintraocular implants.

In some cases, the implants are formed from a single polymer-drugconjugate (i.e., the implants are formed from a polymer-drug conjugatewhich contains the same active agent, hydrophobic polymer segment,branch point (when present), and hydrophilic polymer segment orsegments).

In other embodiments, the implants are formed from a mixture of two ormore different polymer-drug conjugates. For example, implants may beformed from two or more polymer-drug conjugates containing differentactive agents and the same hydrophobic polymer segment, branch point(when present), and hydrophilic polymer segment or segments. Suchimplants can be used, for example, to co-administer two or more activeagents. In other cases, the implants are formed from two or morepolymer-drug conjugates containing the same active agent, and differenthydrophobic polymer segments, branch points (when present), and/orhydrophilic polymer segments. Such implants can be used, for example, tovary the release rate of active agents. The implants can also be formedfrom two or more polymer-drug conjugates containing different activeagents and different hydrophobic polymer segments, branch points (whenpresent), and/or hydrophilic polymer segments.

Implants can also be formed from blends of one or more non-linearmultiblock copolymer-drug conjugates with one or more additionalpolymers. In these cases, the one or more additional polymers can benon-biodegradable or biodegradable polymers, although biodegradablepolymers are preferred. In these embodiments, the identity and quantityof the one or more additional polymers can be selected, for example, toinfluence particle stability, i.e. that time required for distributionto the site where delivery is desired, and the time desired fordelivery.

Representative synthetic polymers which can be blended with non-linearmultiblock copolymer-drug conjugates include poly(hydroxy acids) such aspoly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolicacid), poly(lactide), poly(glycolide), poly(lactide-co-glycolide),polyanhydrides, polyorthoesters, polyamides, polycarbonates,polyalkylenes such as polyethylene and polypropylene, polyalkyleneglycols such as poly(ethylene glycol), polyalkylene oxides such aspoly(ethylene oxide), polyalkylene terepthalates such as poly(ethyleneterephthalate), polyvinyl alcohols, polyvinyl ethers, polyvinyl esters,polyvinyl halides such as poly(vinyl chloride), polyvinylpyrrolidone,polysiloxanes, poly(vinyl alcohols), poly(vinyl acetate), polystyrene,polyurethanes and co-polymers thereof, derivativized celluloses such asalkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, celluloseesters, nitro celluloses, methyl cellulose, ethyl cellulose,hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutylmethyl cellulose, cellulose acetate, cellulose propionate, celluloseacetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, and cellulose sulphate sodium salt (jointlyreferred to herein as “synthetic celluloses”), polymers of acrylic acid,methacrylic acid or copolymers or derivatives thereof including esters,poly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate) (jointly referred to herein as “polyacrylic acids”),poly(butyric acid), poly(valeric acid), andpoly(lactide-co-caprolactone), copolymers and blends thereof. As usedherein, “derivatives” include polymers having substitutions, additionsof chemical groups, for example, alkyl, alkylene, hydroxylations,oxidations, and other modifications routinely made by those skilled inthe art.

Examples of preferred biodegradable polymers include polymers of hydroxyacids such as lactic acid and glycolic acid, and copolymers with PEG,polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid),poly(valeric acid), poly(lactide-co-caprolactone), blends and copolymersthereof.

Examples of preferred natural polymers include proteins such as albuminand prolamines, for example, zein, and polysaccharides such as alginate,cellulose and polyhydroxyalkanoates, for example, polyhydroxybutyrate.The in vivo stability of the implant can be adjusted during theproduction by using polymers such as polylactide-co-glycolidecopolymerized with polyethylene glycol (PEG).

Examples of preferred non-biodegradable polymers include ethylene vinylacetate, poly(meth)acrylic acid, polyamides, copolymers and mixturesthereof

1. Implant Size and Shape

The implants may be of any geometry such as fibers, sheets, films,microspheres, spheres, rods, circular discs, or plaques. Implant size isdetermined by factors such as toleration for the implant, location ofthe implant, size limitations in view of the proposed method of implantinsertion, ease of handling, etc.

Where sheets or films are employed, the sheets or films will be in therange of at least about 0.5 mm×0.5 mm, usually about 3 to 10 mm×5 to 10mm with a thickness of about 0.1 to 1.0 mm for ease of handling. Wherefibers are employed, the fiber diameter will generally be in the rangeof about 0.05 to 3 mm and the fiber length will generally be in therange of about 0.5 to 10 mm.

The size and shape of the implant can also be used to control the rateof release, period of treatment, and drug concentration at the site ofimplantation. Larger implants will deliver a proportionately largerdose, but depending on the surface to mass ratio, may have a slowerrelease rate. The particular size and geometry of the implant are chosento suit the site of implantation.

Intraocular implants may have a size of between about 5 μm and about 2mm, or between about 10 μm and about 1 mm for administration with aneedle, greater than 1 mm, or greater than 2 mm, such as 3 mm or up to10 mm, for administration by surgical implantation. The vitreous chamberin humans is able to accommodate relatively large implants of varyinggeometries, having lengths of, for example, 1 to 10 mm. The implant maybe a cylindrical pellet (e.g., rod) with dimensions of about 2 mm×0.75mm diameter. The implant may be a cylindrical pellet with a length ofabout 7 mm to about 10 mm, and a diameter of about 0.75 mm to about 1.5mm. In certain embodiments, the implant is in the form of an extrudedfilament with a diameter of about 0.5 mm, a length of about 6 mm, and aweight of approximately 1 mg.

Intraocular implants may also be designed to be least somewhat flexibleso as to facilitate both insertion of the implant in the eye, such as inthe vitreous, and subsequent accommodation of the implant. The totalweight of the implant is usually about 250 to 5000 μg, more preferablyabout 500-1000 μg. In certain embodiments, the intraocular implant has amass of about 500 μg, 750 μg, or 1000 μg.

2. Methods of Manufacture

Implants can be manufactured using any suitable technique known in theart. Examples of suitable techniques for the preparation of implantsinclude solvent evaporation methods, phase separation methods,interfacial methods, molding methods, injection molding methods,extrusion methods, coextrusion methods, carver press method, die cuttingmethods, heat compression, and combinations thereof. Suitable methodsfor the manufacture of implants can be selected in view of many factorsincluding the properties of the polymer/polymer segments present in theimplant, the properties of the one or more active agents present in theimplant, and the desired shape and size of the implant. Suitable methodsfor the preparation of implants are described, for example, in U.S. Pat.No. 4,997,652 and U.S. Patent Application Publication No. US2010/0124565.

In certain cases, extrusion methods may be used to avoid the need forsolvents during implant manufacture. When using extrusion methods, thepolymer/polymer segments and active agent are chosen so as to be stableat the temperatures required for manufacturing, usually at least about85 degrees Celsius. However, depending on the nature of the polymericcomponents and the one or more active agents, extrusion methods canemploy temperatures of about 25 degrees Celsius to about 150 degreesCelsius, more preferably about 65 degrees Celsius to about 130 degreesCelsius.

Implants may be coextruded in order to provide a coating covering all orpart of the surface of the implant. Such coatings may be erodible ornon-erodible, and may be impermeable, semi-permeable, or permeable tothe active agents, water, or combinations thereof. Such coatings can beused to further control release of the active agent from the implant.

Compression methods may be used to make the implants. Compressionmethods frequently yield implants with faster release rates thanextrusion methods. Compression methods may employ pressures of about50-150 psi, more preferably about 70-80 psi, even more preferably about76 psi, and use temperatures of about 0 degrees Celsius to about 115degrees Celsius, more preferably about 25 degrees Celsius.

IV. Pharmaceutical Formulations

Pharmaceutical formulations are provided containing particles formedfrom one or more polymer-drug conjugates in combination with one or morepharmaceutically acceptable excipients. Representative excipientsinclude solvents, diluents, pH modifying agents, preservatives,antioxidants, suspending agents, wetting agents, viscosity modifiers,tonicity agents, stabilizing agents, and combinations thereof. Suitablepharmaceutically acceptable excipients are preferably selected frommaterials which are generally recognized as safe (GRAS), and may beadministered to an individual without causing undesirable biologicalside effects or unwanted interactions.

In some cases, the pharmaceutical formulation contains only one type ofpolymer-drug conjugate particles (i.e., the polymer-drug conjugateparticles incorporated into the pharmaceutical formulation have the samecomposition). In other embodiments, the pharmaceutical formulationcontains two or more different types of polymer-drug conjugate particles(i.e., the pharmaceutical formulation contains two or more populationsof polymer-drug conjugate particles, wherein the populations ofpolymer-drug conjugate particles have different chemical compositions,different average particle sizes, and/or different particle sizedistributions).

A. Additional Active Agents

Pharmaceutical compositions can contain one or more additional activeagents which are not present in the polymer-drug conjugate. In somecases, one or more additional active agents may be encapsulated in,dispersed in, or otherwise associated with particles formed from one ormore polymer-drug conjugates. In certain embodiments, one or moreadditional active agents may also be dissolved or suspended in thepharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition contains one ormore local anesthetics. Representative local anesthetics includetetracaine, lidocaine, amethocaine, proparacaine, lignocaine, andbupivacaine. In some cases, one or more additional agents, such as ahyaluronidase enzyme, is also added to the formulation to accelerate andimproves dispersal of the local anesthetic.

B. Excipients

Particles formed from the polymer-drug conjugates will preferably beformulated as a solution or suspension for injection or topicalapplication to the eye. Pharmaceutical formulations for ocularadministration are preferably in the form of a sterile aqueous solutionor suspension of particles formed from one or more polymer-drugconjugates. Acceptable solvents include, for example, water, Ringer'ssolution, phosphate buffered saline (PBS), and isotonic sodium chloridesolution. The formulation may also be a sterile solution, suspension, oremulsion in a nontoxic, parenterally acceptable diluent or solvent suchas 1,3-butanediol.

In some instances, the formulation is distributed or packaged in aliquid form. Alternatively, formulations for ocular administration canbe packed as a solid, obtained, for example by lyophilization of asuitable liquid formulation. The solid can be reconstituted with anappropriate carrier or diluent prior to administration.

Solutions, suspensions, or emulsions for ocular administration may bebuffered with an effective amount of buffer necessary to maintain a pHsuitable for ocular administration. Suitable buffers are well known bythose skilled in the art and some examples of useful buffers areacetate, borate, carbonate, citrate, and phosphate buffers.

Solutions, suspensions, or emulsions for ocular administration may alsocontain one or more tonicity agents to adjust the isotonic range of theformulation. Suitable tonicity agents are well known in the art and someexamples include glycerin, mannitol, sorbitol, sodium chloride, andother electrolytes.

Solutions, suspensions, or emulsions for ocular administration may alsocontain one or more preservatives to prevent bacterial contamination ofthe ophthalmic preparations. Suitable preservatives are known in theart, and include polyhexamethylenebiguanidine (PHMB), benzalkoniumchloride (BAK), stabilized oxychloro complexes (otherwise known asPurite®), phenylmercuric acetate, chlorobutanol, sorbic acid,chlorhexidine, benzyl alcohol, parabens, thimerosal, and mixturesthereof.

Solutions, suspensions, or emulsions for ocular administration may alsocontain one or more excipients known art, such as dispersing agents,wetting agents, and suspending agents.

For other routes of administration, the particles or drug conjugates maybe suspended or emulsified in one or more of the same vehicles as usedfor ocular administration. These can be administered by injection, drop,spray, topical application, or depo, to a mucosal surface such as theeye (ocular), nose (nasal), mouth (buccal), rectum, vagina, orally, orinjection into the bloodstream, tissue or skin.

V. Methods of Use

In certain embodiments, pharmaceutical compositions containing particlesformed from one or more of the polymer-drug conjugates are used to treator prevent one or more diseases of the eye. In some embodiments,implants formed from one or more of the polymer-drug conjugates are usedto treat or prevent one or more diseases of the eye.

When administered to the eye, the particles and implants release a lowdose of one or more active agents over an extended period of time,preferably longer than 3 days, more preferably longer than 7 days, mostpreferably longer than ten days. In some embodiments, the particles andimplants release an effective amount of one or more active agents over aperiod of seven days to 24 weeks, more preferably from seven days toeight weeks, preferably from seven days to three weeks. In other cases,the particles and implants release an effective amount of one or moreactive agents over a period of seven days to 2 years, more preferablyfrom seven days to 56 weeks, more preferably from four weeks to 56weeks, most preferably from eight weeks to 28 weeks.

The structure of the polymer-drug conjugate, particle/implantmorphology, dosage of particles, and the amount of polymer-drugconjugate incorporated in the particle/implant can be tailored toadminister a therapeutically effective amount of one or more activeagents to the eye over an extended period of time while minimizing sideeffects, such as the reduction of scoptopic ERG b-wave amplitudes and/orretinal degeneration.

A. Ocular Diseases and Disorders to be Treated

Pharmaceutical compositions containing particles formed from one or moreof the polymer-drug conjugates provided herein are administered to theeye of a patient in need thereof to treat or prevent one or morediseases or disorders of the eye. Implants formed from one or more ofthe polymer-drug conjugates can also be administered to the eye of apatient in need thereof to treat or prevent one or more diseases ordisorders of the eye.

In some cases, the disease or disorder of the eye affects the posteriorsegment of the eye. The posterior segment of the eye, as used herein,refers to the back two-thirds of the eye, including the anterior hyaloidmembrane and all of the optical structures behind it, such as thevitreous humor, retina, choroid, and optic nerve.

In preferred embodiments, a pharmaceutical composition containingparticles formed from one or more of the polymer-drug conjugatesprovided herein is administered to treat or prevent an intraocularneovascular disease. In certain embodiments, the particles are formedfrom a polymer-drug conjugate containing an anthracycline, such asdaunorubicin or doxorubicin, which inhibits smooth muscle cellproliferation.

Eye diseases, particularly those characterized by ocularneovascularization, represent a significant public health concern.Intraocular neovascular diseases are characterized by unchecked vasculargrowth in one or more regions of the eye. Unchecked, the vascularizationdamages and/or obscures one or more structures in the eye, resulting invision loss. Intraocular neovascular diseases include proliferativeretinopathies, choroidal neovascularization (CNV), age-related maculardegeneration (AMD), diabetic and other ischemia-related retinopathies,diabetic macular edema, pathological myopia, von Hippel-Lindau disease,histoplasmosis of the eye, central retinal vein occlusion (CRVO),corneal neovascularization, and retinal neovascularization (RNV).Intraocular neovascular diseases afflict millions worldwide, in manycases leading to severe vision loss and a decrease in quality of lifeand productivity.

Other diseases and disorders of the eye, such as uveitis, are alsodifficult to treat using existing therapies. Uveitis is a general termreferring to inflammation of any component of the uveal tract, such asthe iris, ciliary body, or choroid. Inflammation of the overlyingretina, called retinitis, or of the optic nerve, called optic neuritis,may occur with or without accompanying uveitis.

Ocular complications of uveitis may produce profound and irreversibleloss of vision, especially when unrecognized or treated improperly. Themost frequent complications of uveitis include retinal detachment,neovascularization of the retina, optic nerve, or iris, and cystoidmacular edema. Macular edema (ME) can occur if the swelling, leaking,and background diabetic retinopathy (BDR) occur within the macula, thecentral 5% of the retina most critical to vision. ME is a common causeof severe visual impairment.

The neovascularization can be caused by a tumor. The tumor may be eithera benign or malignant tumor. Exemplary benign tumors include hamartomasand neurofibromas. Exemplary malignant tumors include choroidalmelanoma, uveal melanoma or the iris, uveal melanoma of the ciliarybody, retinoblastoma, or metastatic disease (e.g., choroidalmetastasis).

The neovascularization may be associated with an ocular wound. Forexample, the wound may the result of a traumatic injury to the globe,such as a corneal laceration or ophthalmic surgery.

The polymer-drug conjugates can be administered to prevent or reduce therisk of proliferative vitreoretinopathy following vitreoretinal surgery,prevent corneal haze following corneal surgery (such as cornealtransplantation and eximer laser surgery), prevent closure of atrabeculectomy, or to prevent or substantially slow the recurrence ofpterygii.

The polymer-drug conjugates can be administered to treat or prevent aneye disease associated with inflammation. In such cases, thepolymer-drug conjugate preferably contains an anti-inflammatory agent.Exemplary inflammatory eye diseases include, but are not limited to,uveitis, endophthalmitis, and ophthalmic trauma or surgery. The eyedisease may also be an infectious eye disease, such as HIV retinopathy,toxocariasis, toxoplasmosis, and endophthalmitis.

Pharmaceutical compositions containing particles formed from one or moreof the polymer-drug conjugates can also be used to treat or prevent oneor more diseases that affect other parts of the eye, such as dry eye,meibomitis, glaucoma, conjunctivitis (e.g., allergic conjunctivitis,vernal conjunctivitis, giant papillary conjunctivitis, atopickeratoconjunctivitis), neovascular glaucoma with irisneovascularization, and iritis.

The compositions and implants are useful for treatment of otherdisorders, based on the selection of the active agent and the route ofadministration. Although there are benefits achieved via the ocularroute, including extended efficacy and alleviation of inflammation, theconjugates should also provide benefits and modified pharmacokineticswhen administered via another route.

B. Methods of Administration

1. Mode of Administration

The polymer-drug conjugates can be administered locally to the eye byintravitreal injection (e.g., front, mid or back vitreal injection),subconjunctival injection, intracameral injection, injection into theanterior chamber via the temporal limbus, intrastromal injection,intracorneal injection, subretinal injection, and intraocular injection.In a preferred embodiment, the pharmaceutical composition isadministered by intravitreal injection.

Alternatively, pharmaceutical compositions containing particles formedfrom one or more polymer-drug conjugates can be administered via eyedrops applied to the surface of the cornea.

For other routes of administration, the particles or drug conjugates maybe suspended or emulsified in one or more of the same vehicles as usedfor ocular administration. These can be administered by injection, drop,spray, topical application, or depo, to a mucosal surface such as theeye (ocular), nose (nasal), mouth (buccal), rectum, vagina, orally, orinjection into the bloodstream, tissue or skin.

The implants described herein can be administered to the eye usingsuitable methods for implantation known in the art. In certainembodiments, the implants are injected intravitreally using a needle,such as a 22-gauge needle. Placement of the implant intravitreally maybe varied in view of the implant size, implant shape, and the disease ordisorder to be treated.

In some embodiments, a pharmaceutical composition containing particlesformed from one or more of the polymer-drug conjugates areco-administered with one or more additional active agents.“Co-administration”, as used herein, refers to administration of thepolymer-drug conjugates and one or more additional active agents withinthe same dosage form, as well as administration of the polymer-drugconjugates and one or more additional active agents using differentdosage forms simultaneously or as essentially the same time.“Essentially at the same time” as used herein generally means within tenminutes, preferably within five minutes, more preferably within twominutes, most preferably within in one minute.

2. Dosage

Preferably, the particles and implants formed from the polymer-drugconjugates will release an effective amount of one or more therapeuticagent over an extended period of time. In preferred embodiments, theparticles and implants release an effective amount of one or more activeagents over a period of at least two weeks, more preferably over aperiod of at least four weeks, more preferably over a period of at leastsix weeks, most preferably over a period of at least eight weeks. Insome embodiments, the particles and implants release an effective amountof one or more active agents over a period of three months or longer.

In some cases, a pharmaceutical formulation containing particles formedfrom one or more polymer-drug conjugates (or an implant formed from oneor more polymer-drug conjugates) is administered to a patient in needthereof in a therapeutically effective amount to decrease choroidalneovascularization. In some embodiments, a pharmaceutical formulationcontaining particles formed from one or more polymer-drug conjugates (oran implant formed from one or more polymer-drug conjugates) isadministered to a patient in need thereof in a therapeutically effectiveamount to decrease the area of CNV, as measured by fluoresceinangiography, by at least 15%, more preferably at least 25%, morepreferably at least 40%, most preferably at least 50%.

In some cases, a pharmaceutical formulation containing particles formedfrom one or more polymer-drug conjugates (or an implant formed from oneor more polymer-drug conjugates) is administered to a patient in needthereof in a therapeutically effective amount to decrease retinalneovascularization. In some cases, a pharmaceutical formulation (or animplant formed from one or more polymer-drug conjugates) is administeredto a patient in need thereof in a therapeutically effective amount todecrease the area of RNV, as measured by fluorescein angiography, by atleast 15%, more preferably at least 25%, more preferably at least 40%,most preferably at least 50%.

An effective dosage can be determined by one of skill in the art basedon the known therapeutic efficacy of the drug which is attached to thepolymer and determining the pharmacokinetics of the conjugate.

3. Therapeutic Efficacy

In the case of age-related macular degeneration, therapeutic efficacy ina patient can be measured by one or more of the following: assessing themean change in the best corrected visual acuity (BCVA) from baseline toa desired time, assessing the proportion of patients who lose fewer than15 letters (3 lines) in visual acuity at a desired time as compared to abaseline, assessing the proportion of patients who gain greater than orequal to 15 letters (3 lines) in visual acuity at a desired time ascompared to a baseline, assessing the proportion of patients with avisual acuity Snellen equivalent of 20/2000 or worse at a desired time,assessing the National Eye Institute Visual Functioning Questionnaire,and assessing the size of CNV and the amount of leakage of CNV at adesired time using fluorescein angiography.

In certain embodiments, at least 25%, more preferably at least 30%, morepreferably at least 35%, most preferably at least 40% of the patientswith recent onset CNV who are treated with the formulations describedherein improve by three or more lines of vision.

For other diseases or disorders, efficacy is determined based on aremission or reduction in one or more symptoms of the disease ordisorder. For example, in the case of using a drug such as rapamycin tolimit uncontrolled proliferation of muscle cells following angioplasty,one would start with a dosage comparable to rapamycin without thepolymer, the assess efficacy with this dosage with the polymer, withefficacy being correlated with a decrease in restenosis relative tocontrol.

In the case of treatment of tumors, efficacy can be measured as adecrease in the rate of proliferation, decrease in tumor mass, ordecrease in metastasis.

The present invention will be further understood by reference to thefollowing non-limiting examples.

Example 1: Preparation of Polyanhydride-Drug Conjugate ParticlesSynthesis of Polymers

(Polyethylene glycol)₃-co-poly(sebacic acid) (PEG₃-PSA) was prepared bymelt polycondensation. Briefly, sebacic acid was refluxed in aceticanhydride to form a sebacic acid prepolymer (Acyl-SA).Citric-Polyethylene glycol (PEG₃) was prepared using methods known inthe art (Ben-Shabat, S. et al. Macromol. Biosci. 6:1019-1025 (2006)).2.0 g of CH₃O-PEG-NH₂, 26 mg of citric acid, 83 mg ofdicyclohexylcarbodiimide (DCC), and 4.0 mg of 4-(dimethylamino)pyridine(DMAP) were added to 10 mL of methylene chloride. This mixture wasstirred overnight at room temperature, then precipitated, washed withether, and dried under vacuum to isolate PEG₃. Next, Acyl-SA (90% w/w)and PEG₃ (10% w/w) were polymerized at 180° C. for 30 minutes. Nitrogengas was swept into the flask for 30 seconds every 15 minutes.

The reaction was allowed to proceed for 30 min. Polymers were cooled toambient temperature, dissolved in chloroform, and precipitated intoexcess petroleum ether. The precipitate was collected by filtration anddried under vacuum to constant weight.

Formation of DXR-PSA-PEG₃ Nanoparticles

DXR-PSA-PEG₃ nanoparticles were prepared by dissolving PEG₃-PSA with DXRat defined ratios in 3 mL dichloromethane and lmL DMSO and reacting for2 hrs at 50° before homogenizing (L4RT, Silverson Machines, EastLongmeadow, Mass.) into 100 mL of an aqueous solution containing 1%polyvinyl alcohol (25 kDa, Sigma). Particles were then hardened byallowing chloroform to evaporate at room temperature while stirring for2 hours. The particles were collected by centrifugation (20,000×g for 20min at 4° C.), and washed thrice with double distilled water. Particlesize was determined by dynamic light scattering using a ZetaSizer NanoZS (Malvern Instruments, Southborough, Mass.). Size measurements wereperformed at 25° C. at a scattering angle of 90°.

DXR Release from DXR-PSA-PEG₃ Nanoparticles In Vitro

DXR-PSA-PEG₃ nanoparticles were suspended in phosphate buffered saline(PBS, pH 7.4) at 2 mg/mL and incubated at 37° C. on a rotating platform(140 RPM). At selected time points, supernatant was collected bycentrifugation (13,500×g for 5 min) and particles were resuspended infresh PBS. DXR content was measured by absorbance at 480 nm.

Results

The DXR-PSA-PEG₃ nanoparticles prepared above contained 23.6% DXR (byweight), and had an average particle size of 647 nm. In vitro studiesshowed that DXR was released from the nanoparticles as a conjugate withsebacic acid in a steady fashion for up to two weeks under sinkconditions in PBS at 37° C. with no initial rapid drug release phase(i.e., no “burst effect”).

Example 2: Treatment of Choroidal Neovascularization in a Mouse Model ofCNV

Materials and Methods

Pathogen-free C57BL/6 mice (Charles River, Wilmington, Mass.) weretreated in accordance with the Association for Research in Vision andOphthalmology Statement for the Use of Animals in Ophthalmic and VisionResearch and the guidelines of the Johns Hopkins University Animal Careand Use Committee.

Choroidal NV was induced by laser photocoagulation-induced rupture ofBruch's membrane as previously described (Tobe, T. et al., Am. J.Pathol. 135(5): 1641-1646(1998)). Briefly, 5-6-week-old female C57BL/6mice were anesthetized with ketamine hydrochloride (100 mg/kg bodyweight) and pupils were dilated. Laser photocoagulation (75 μm spotsize, 0.1 sec duration, 120 mW) was performed in the 9, 12, and 3o'clock positions of the posterior pole of each eye with the slit lampdelivery system of an OcuLight GL diode laser (Iridex, Mountain View,Calif.) and a handheld cover slip as a contact lens to view the retina.Production of a bubble at the time of laser, which indicates rupture ofBruch's membrane, is an important factor in obtaining CNV; therefore,only burns in which a bubble was produced were included in the study.

Immediately after laser-induced rupture of Bruch's membrane, mice wererandomized to various treatment groups for intraocular injections.Intravitreal injections were done under a dissecting microscope with aHarvard Pump Microinjection System and pulled glass micropipettes.

At 1, 4, 7, and 14 days after injection, fundus photographs were takenwith a Micron III® camera (Phoenix Research Laboratories Inc.,Pleasanton, Calif.). After 14 days, the mice were perfused with 1 ml ofPBS containing 25 mg/ml of fluorescein-labeled dextran (2×10⁶ Daltonsaverage molecular weight; Sigma-Aldrich, St. Louis, Mo.) and choroidalflat mounts were examined by fluorescence microscopy. Images werecaptured with a Nikon Digital Still Camera DXM1200 (Nikon InstrumentsInc., New York, N.Y.). Image analysis software (Image-Pro® Plus; MediaCybernetics, Silver Spring, Md.) was used to measure the total area ofCNV at each rupture site with the investigator masked with respect totreatment group.

Treatment of Oxygen-Induced Ischemic Retinopathy

C57BL/6 mice placed in 75% oxygen at postnatal day (P) 7 and at P12 werereturned to room air and given an intraocular injection of PBS or PBScontaining Daunorubicin, Doxorubicin, or DXR-PSA-PEG₃ nanoparticles. AtP17, the area of retinal NV on the surface of the retina was measured.Briefly, P17 mice were given an intraocular injection of 1 μl of ratanti-mouse platelet endothelial cell adhesion molecule-1 (PECAM-1)antibody (Pharmingen, San Jose, Calif.) and after 12 hours they wereeuthanized and eyes were fixed in PBS-buffered formalin for 5 hours atroom temperature. Retinas were dissected, washed, and incubated withgoat-anti rat polyclonal antibody conjugated with Alexa 488 (Invitrogen,Carlsbad, Calif.) at 1:500 dilution at room temperature for 45 minutesand flat mounted. An observer masked with respect to treatment groupmeasured the area of NV per retina by image analysis.

Treatment of VEGF-Induced Retinal Neovascularization

Hemizygous rhodopsin/VEGF transgenic mice that express VEGF inphotoreceptors were given an intraocular injection of 1 μl of PBS or PBScontaining 10 μg DXR-PSA-PEG₃ nanoparticles at P14. At P21, P28, P35,P42 or P49, the mice were anesthetized, perfused withfluorescein-labeled dextran (2×10⁶ average molecular weight,Sigma*-Aldrich), and retinal flat mounts were examined by fluorescencemicroscopy (Axioskop2 plus; Zeiss, Thornwood, N.Y.) at 400×magnification, which provides a narrow depth of field, so that whenneovascularization along the outer edge of the retina is brought intofocus, the remainder of the retinal vessels are out of focus, allowingeasy delineation and quantification of the neovascularization. Imageswere digitized with a three-color charge-coupled device video camera(Cool SNAP™-Pro; Media Cybernetics, Silver Spring, Md.) and a framegrabber. Image analysis software (Image-Pro Plus 5.0; Media Cybernetics,Silver Spring, Md.) was set to recognize fluorescently stainedneovascularization and used to calculate the total area ofneovascularization per retina. The investigator performing imageanalysis was masked with respect to treatment group.

Recording of Electroretinograms (ERGs)

Adult C57BL/6 mice were given an intraocular injection of 1 μl of PBS orPBS containing of 0.1, 1.0, or 10 μg of Daunorubicin or Doxorubicin, or1.0 or 10 μg DXR-PSA-PEG₃ nanoparticles. Scotopic and photopic ERGs wererecorded at one, seven and 14 days after injection using an Espion ERGDiagnosys machine. For scotopic recordings, mice were dark adaptedovernight, and for photopic recordings, mice were adapted for 10 min tobackground white light at an intensity of 30 cd/m². The mice wereanesthetized with an intraperitoneal injection of ketamine hydrochloride(100 mg/kg body weight) and xylazine (5 mg/kg body weight). Pupils weredilated with Midrin P containing of 0.5% tropicamide and 0.5%phenylephrine, hydrochloride (Santen Pharmaceutical Co., Osaka, Japan).The mice were placed on a pad heated to 39° C. and platinum loopelectrodes were placed on each cornea after application of Gonioscopicprism solution (Alcon Labs, Fort Worth, Tex.). A reference electrode wasplaced subcutaneously in the anterior scalp between the eyes and aground electrode was inserted into the tail. The head of the mouse washeld in a standardized position in a ganzfeld bowl illuminator thatensured equal illumination of the eyes. Recordings for both eyes weremade simultaneously with electrical impedance balanced. Scotopic ERGswere recorded at 11 intensity levels of white light ranging from −3.00to 1.40 log cd-s/m2. Six measurements were averaged for each flashintensity. Photopic ERGs were recorded at three intensity levels ofwhite light ranging from 0.60 to 1.40 log cd-s/m2 with a 30 cd/m2background. Five measurements were averaged for each flash intensity.

Measurement of Outer Nuclear Layer (ONL) Thickness

ONL thickness was measured. Adult C57BL/6 mice were given an intraocularinjection of 1 μl of PBS or PBS containing of 0.1, 1.0, or 10 μg ofDaunorubicin or Doxorubicin, or 1.0 or 10 μg DXR-PSA-PEG₃ nanoparticles.Mice were euthanized, a mark was placed at 12:00 at the corneal limbus,and eyes were removed and embedded in optimal cutting temperaturecompound. Ten micrometer frozen sections were cut parallel to the 12:00or 9:00 meridian through the optic nerve and fixed in 4%paraformaldehyde. The sections were stained with hematoxylin and eosin,examined with an Axioskop microscope (Zeiss, Thornwood, N.Y.), andimages were digitized using a three charge-coupled device (CCD) colorvideo camera (IK-TU40A; Toshiba, Tokyo, Japan) and a frame grabber.Image-Pro Plus software (Media Cybernetics, Silver Spring, Md.) was usedto outline the ONL. With the observer masked with respect to treatmentgroup, ONL thickness was measured at six locations, 25% (S1), 50% (S2),and 75% (S3) of the distance between the superior pole and the opticnerve and 25% (I1), 50% (I2), and 75% (I3) of the distance between theinferior pole the optic nerve.

Statistical Analysis

Data were expressed as mean±SEM. Statistical analysis was performedusing Student's t-test and P<0.05 was considered significant.

Results

Anthracyclines Suppress Choroidal and Retinal NV

In a mouse model of choroidal NV (Tobe, T. et al. Am. J. Pathol.153:1641-1646 (1998)) that is predictive of drug effects in patientswith neovascular AMD (Saishin, Y. et al. J. Cell Physiol. 195:241-248(2003)), intraocular injection of 10 μg of DNR suppressed choroidal NV,while injection of 1 or 0.1 μg had no significant effect (FIG. 1A).Similarly, intraocular injection of 10 μg of DXR suppressed choroidal NVand injections of 1 or 0.1 μg did not have a significant effect (FIG.1B).

In neonatal mice with oxygen-induced ischemic retinopathy, a modelpredictive of effects in proliferative diabetic retinopathy, intraocularinjection of 1 μg of DNR markedly reduced the area of retinal NV, 0.1 μgcaused a small reduction, and 0.01 μg had no significant effect (FIG.2A). The NV was visualized on retinal flat mounts after in vivoimmunofluorescent staining with anti-PECAM1, a technique thatselectively stains NV and hyaloid vessels. Intraocular injection of 1 μgof DXR, but not 0.1 or 0.01 μg, significantly reduced the area ofretinal NV (FIG. 2B). Five days after injection of 1 μg of DNR or DXR,precipitated drug was visualized on the surface of the retina. The meanarea of choroidal or retinal NV in fellow eyes was not significantlydifferent from that in eyes of mice in which both eyes were injectedwith vehicle indicating that there was no systemic effect fromintraocular injections of DNR or DXR.

Effect of Intraocular Injections of DNR or DXR on Retinal Function

Since DNR and DXR are antimetabolites as well as HIF-1 inhibitors weexamined their effect on retinal function assessed by ERGs. Fourteendays after intraocular injections of 1 μg, but not 0.1 μg, of DNR or DXRthere was a significant reduction in mean scotopic and photopic b-waveamplitudes. These data indicate that while DNR and DXR strongly suppressocular NV, bolus injections of free drugs can cause retinal toxicity.

Retinal Toxicity after Intraocular Injection of Digoxin

It has been previously demonstrated that intraocular injections of0.01-0.25 μg of digoxin suppress HIF-1 transcriptional activity andocular NV (Yoshida, T. et al. FASEB J. 24:1759-1767 (2010)). To explorewhether the deleterious effects of DXR and DNR on retinal function mightbe related to their suppression of HIF-1 activity, the effects ofintraocular injection of 0.25 and 0.05 μg of digoxin on retinal functionwere measured. One week after intraocular injection of 0.25 μg ofdigoxin, there was a significant reduction in mean scotopic a-waveamplitude, mean scotopic b-wave amplitude, and mean photopic b-waveamplitude. There was also a reduction in outer nuclear layer thicknessat 3 of 6 measurement locations in the retina, indicating death ofphotoreceptor cells. These results are consistent with substantialtoxicity after intraocular injection of 0.25 μg of digoxin. Intraocularinjection of 0.05 μg of digoxin was less toxic, but still causedsignificant reduction in mean scotopic and photopic b-wave amplitudes.Thus, for both anthracyclines and digoxin, injection of free drug intothe eye carries risk of retinal toxicity.

Effect of DXR Polymer Nanoparticles on Ocular NV

The effect of intraocular injection of DXR nanoparticles was firsttested in mice with laser-induced choroidal NV. After laser-inducedrupture of Bruch's membrane, C57BL/6 mice received an intraocularinjection of 10, 1.0, or 0.1 μg of DXR-PSA-PEG₃ nanoparticles. Fundusphotos of the animals that received 1 μg of DXR-PSA-PEG₃ nanoparticlesshowed a large orange mass of nanoparticles overlying the posteriorretina 1 day after injection that decreased slowly over time and wasstill readily visible on day 14. Particles remained visible over periodsof time as long as five weeks.

In mice perfused with fluorescein-labeled dextran to visualize choroidalNV by fluorescence microscopy at day 14, the area of choroidal NVappeared smaller in eyes given an intraocular injection of DXR-PSA-PEG₃nanoparticles compared to fellow eyes injected with PBS. Image analysisconfirmed that compared to eyes injected with PBS, the mean area ofchoroidal NV was significantly less in eyes injected with 10, 1, or 0.1μg of DXR-PSA-PEG₃ nanoparticles (FIG. 3A).

The effect of DXR-PSA-PEG₃ nanoparticles on already establishedchoroidal NV was investigated by allowing the NV to grow for 7 days andthen injecting 1 μg of DXR-PSA-PEG₃ nanoparticles. Seven days afterinjection, eyes injected with DXR-PSA-PEG₃ nanoparticles had a mean areaof choroidal NV that was significantly less than that seen in controleyes injected with PBS, and also significantly less than the baselineamount of choroidal NV that was present at 7 days (FIG. 3B). Thisindicates that DXR-PSA-PEG₃ nanoparticles cause regression ofestablished choroidal NV.

The DXR-PSA-PEG₃ nanoparticle formulation was also investigated using amodel of ischemia-induced retinal neovascularization (Smith, L. E. H. etal. Invest. Ophthalmol. Vis. Sci. 35:101-111 (1994)). Intraocularinjections of 1 μg of DXR-PSA-PEG₃ nanoparticles significantly reducedthe mean area of retinal NV compared to fellow eyes injected with PBS(FIG. 4 ).

Prolonged Suppression of NV after Intraocular Injection of DXR PolymerNanoparticles in Rho/VEGF Transgenic Mice

Rho/VEGF transgenic mice, in which the rhodopsin promoter drivesexpression of VEGF in photoreceptors, have sustained expression of VEGFstarting at postnatal day (P) 7, and provide an excellent model to testthe duration of activity of a therapeutic agent (Okamoto, N. et. al. Am.J. Pathol. 151:281-291 (1997)).

At P14, hemizygous rho/VEGF mice were given an intraocular injection of10 μg of DXR-PSA-PEG₃ nanoparticles in one eye and PBS in the felloweye. At 4 (FIG. 5A) or 5 weeks (FIG. 5B) after injection the mean areaof subretinal NV was significantly less in DXR nanoparticle-injectedeyes than vehicle-injected fellow eyes.

Intraocular Injection of 1 or 10 μg of DXR Nanoparticles Did not CauseToxicity as Measured by ERG or ONL Thickness

At 14 days after intraocular injection of 10 μg of DXR-PSA-PEG₃, therewas no significant difference in scotopic or photopic b-wave amplitudescompared to PBS-injected eyes. There was also no difference in outernuclear layer thickness indicating that DXR nanoparticles did not causephotoreceptor cell death.

Example 3. Pharmacokinetic Study in Rabbits

Materials and Methods

Preparation of the PEG₃-PSA Polymer

(Polyethylene glycol)3-co-poly(sebacic acid)(PEG₃-PSA) was synthesizedby melt condensation. Briefly, sebacic acid was refluxed in aceticanhydride to form sebacic acid prepolymer (Acyl-SA). Polyethylene glycol(PEG₃) was prepared by mixing CH₃O-PEG-NH₂ (2.0 g), citric acid (26 g),dicyclohexylcarbodiimide (DCC; 83 mg) and 4-(dimethylamino)pyridine(DMAP, 4.0 mg) which were added to 10 mL methylene chloride, stirredovernight at room temperature, then precipitated and washed with ether,and dried under vacuum. Next, Acyl-SA (90% w/w) and PEG₃ (10% w/w) werepolymerized at 180° C. for 30 minutes. Nitrogen gas was swept into theflask for 30 seconds every 15 minutes.

Polymers were cooled to ambient temperature, dissolved in chloroform andprecipitated into excess petroleum ether. The precipitate was collectedby filtration and dried under vacuum to constant weight, to produce thePEG₃-PSA polymer.

Preparation of the DXR-PSA-PEG₃ Microparticles and Nanoparticles

To prepare DXR-PSA-PEG₃ nanoparticles, 80 mg PEG₃-PSA was dissolved in 6mL dichloromethane (DCM) and 20 mg doxorubicin hydrochloride (DXR)(NetQem LLC, Durham, N.C.) was dissolved in 2 mL dimethylsulfoxide(DMSO). The solutions of polymer and drug were mixed and kept at 50° C.for 30 min. The resulting mixture was homogenized in 50 mL of 1%polyvinyl alcohol (PVA) solution (25 kDa, Polyscience, Niles, Ill.) at10,000 rpm for 3 min using a L4RT homogenizer (Silverson Machines, EastLongmeadow, Mass.). The particle suspension was stirred at roomtemperature for 2 hours to remove dichloromethane. The particles werecollected by centrifugation (20,000×g for 20 minutes at 4° C.) andwashed thrice with ultrapure water prior to lyophilization.

DXR-PSA-PEG₃ microparticles were prepared in a similar fashion. Briefly,200 mg PEG₃-PSA was dissolved in 3 mL DCM and mixed with 40 mg DXRdissolved in 1.5 mL DMSO. Following incubation at 50° C. for 30 min, themixture was homogenized in 100 mL of PVA at 3,000 rpm for 1 min. Afterstirring for 2 hr, particles were collected by centrifugation (9,000×gfor 25 minutes) and washed thrice before lyophilization.

Particle Characterization

Particle size was determined using a Coulter Multisizer IV(Beckman-Coulter Inc., Fullerton, Calif.). Greater than 100,000particles were sized for each batch of microparticles to determine themean particle diameter. Particle morphology was characterized byscanning electron microscopy (SEM) using a cold cathode field emissionSEM (JEOL JSM-6700F, Peabody, Mass.). Drug loading was determined bydissolving dry powder of the particles in DCM and DMSO and theabsorbance was measured using a UV spectrophotometer at 490 nm.

Animal Procedures

Pigmented, Dutch-Belted rabbits were used for these studies (n=10).Animals were treated in accordance with the Association for Research inVision and Ophthalmology Statement of Use of Animals in Ophthalmic andResearch and the guidelines of the Johns Hopkins University Animal Careand Use Committee. For intraocular injections and collection of aqueoushumor, animals were anesthetized with an intramuscular injection ofketamine (25 mg/kg) and xylazine (2.5 mg/Kg). When sedated, the pupilswere dilated with 2.5% phenylephrine hydrochloride and 10% tropicamide.Ocular surface anesthesia was performed using topical instillation of0.5% proparacaine hydrochloride.

For the injections, a 26-gauge needle was carefully introduced into thevitreous cavity, 1.5 mm posterior to the superotemporal limbus, with theneedle tip directed into the mid-vitreous. A volume of 0.1 mL ofDXR-PSA-PEG₃ micro or nanoparticle suspension was delivered to the righteyes, and 0.1 mL vehicle (PBS) was delivered to the left eyes. Theneedle was held in place for 10 seconds before withdrawal to preventreflux from the entry site. Animals were returned to their cages andmonitored until anesthesia was reversed.

At the indicated times, aqueous humor was withdrawn (˜0.1 mL) byinserting a 30-gauge needle through the limbus and removing the aqueoushumor. The samples were stored at −80° C. until use. At the end of thestudy (Day 105 for the nanoparticle-treated animals and Day 115 for themicroparticle-treated animals), animals were euthanized using apentobarbital-based euthanasia (>150 mg/Kg). Animals were enucleated andvitreous was isolated and stored at −80° C. until use.

HPLC Quantitation of Released Drug Conjugates in Rabbit Aqueous Humorand Vitreous Samples

Prior to quantitation of the drug content by HPLC, 100 μL of aqueoushumor sample or vitreous sample was mixed with 200 μL of methanol andincubated at 4° C. for 3 hr. After centrifugation (15,000×g, 10 min) andfiltration through a 0.2 μm PTFE filter, 150 μL of the filtrate wasinjected into a Waters HPLC system equipped with a c18 reverse phasecolumn (5 μm, 4.6×250 mm; Grace, Deerfield Ill.). Released drugconjugate was eluted by an isocratic mobile phase containing water andacetonitrile (60%:40%, v/v) at 1 mL/min and detected using afluorescence detector (excitation wavelength: 500 nm, emissionwavelength: 551 nm). The estimated limit of detection was 10 ng/mL or 20nM. A series of DXR aqueous solutions at different concentrations wereused as calibration standards. The data was analyzed using Empower 3chromatography data software (Waters Corporation, Milford Mass.).

Results

DXR-PSA-PEG₃ Microparticles and Nanoparticles

Microparticles and nanoparticles composed of the DXR-SA-PEG₃ conjugatewere synthesized and characterized as described. Particles were sizedprior to lyophilization and following reconstitution in vehicle (PBS).The microparticles displayed a mean size of 27.2+1.0 um, and thenanoparticles, 0.98+0.02 um prior to lyophilization (Table 1; FIG. 6 ).The average drug loading of the microparticles was 13% and thenanoparticles was 20% (Table 1).

TABLE 1 Characterization of DXR-PSA-PEG₃ Micro and NanoparticlesParticle Diameter by Volume Average Sample ID Post- Drug Prior toreconstitution Loading Type lyophilization in PBS (w/w) Microparticles27.2 ± 10.4 μm 24.3 ± 8.3 μm 13% Nanoparticles 0.98 ± 0.74 μm  3.7 ± 2.0μm 20%

SEM analyses demonstrated discrete particles of the expected size. FIGS.6A and 6B show the size distribution by volume of the microparticles andnanoparticles, respectively.

Duration of Drug Release Following IVT Administration to Rabbits

Rabbits received an intravitreal injection (0.1 mL) of the DXR-PSA-PEG₃microparticles or nanoparticles into their right eyes and vehicle alone(PBS) into their left eyes. At the indicated times, aqueous humor wascollected (˜0.1 mL) and analyzed for the presence of released drugconjugate using a quantitative HPLC-based assay. On Day 115(microparticle group) or Day 105 (nanoparticle group), animals wereeuthanized and aqueous humor and vitreous was collected.

The released drug levels in the AH were compared to that in the vitreousfor each animal.

All rabbits displayed sustained drug release following intravitrealparticle administration (FIG. 7A, Table 2).

TABLE 2 Pharmacokinetics of Intravitreal Delivery of DXR-PSA-PEG₃Particles to Rabbits. DXR-PSA-PEG₃ Microparticles DXR-PSA-PEG₃Nanoparticles DXR Conc. in DXR Conc. in Day Aqueous Humor Day AqueousHumor 1 4.74 ± 2.23 μg/mL 1 6.91 ± 2.40 μg/mL 7 3.45 ± 1.76 μg/mL 8 2.61± 1.11 μg/mL 17 1.63 + 0.65 μg/mL 18 1.51 ± 0.77 μg/mL 31 0.78 ± 0.52μg/mL 33 0.75 ± 0.41 μg/mL 64 0.16 ± 0.21 μg/mL 57 0.37 ± 0.27 μg/mL 920.21 ± 0.45 μg/mL 97 0.22 ± 0.26 μg/mL 115 0.05 ± 0.08 μg/mL 105 0.13 ±0.18 μg/mL

Data presented as mean±SD.

Levels well above the limit of quantitation of the HPLC assay (10 ng/mLor 20 nM) were observed in both the microparticle andnanoparticle-treated animals for the duration of the study, 115 and 105days, respectively (FIG. 7A). Direct comparison of the released druglevels in the AH compared to the vitreous revealed that vitreous levelswere significantly higher than those measured in the AH, up to 188 timeshigher in the vitreous compared to the AH (Table 3, FIG. 7B). The meanreleased drug levels for the microparticle-treated animals at Day 115were 0.09+0.13 uM in the AH and 7.12+12.92 uM in the vitreous. For thenanoparticle-treated animals at Day 105 mean released drug levels were0.23+0.31 uM in the AH and 11.91+10.45 uM in the vitreous (Table 3).Drug levels in the vitreous were 77-90 times higher than drug levelsmeasured in the AH.

FIG. 7A is a graph showing the amount of released DXR drug conjugate(nM) as a function of time (days) in the aqueous humor (AH) of rabbitstreated with microparticles and nanoparticles injected into thevitreous. FIG. 7B is a bar graph comparing the released drug amounts inthe aqueous humor (AH) and vitreous of nanoparticle andmicroparticle-treated rabbits at days 105 and 115, respectively.

TABLE 3 Comparison of Released Drug Levels in the Aqueous Humor vsVitreous Ratio DXR concentration (uM) Treat- Aqueous Humor VitreousVitreous/ ment ug/mL uM ug/mL uM Aqueous Micro- particles Rabbit 1 0.0200.034 0.34 0.59 17 Rabbit 2 0.184 0.317 17.50 30.17 95 Rabbit 3 0.0140.024 1.75 3.01 125 Rabbit 4 0.030 0.052 0.79 1.37 27 Rabbit 5 0.0020.004 0.27 0.47 122 Mean 0.05 ± 0.08 0.09 ± 0.13 4.13 ± 7.50  7.12 ±12.92 77 ± 52 Nano- particles Rabbit 1 0.10 0.17 4.81 8.30 50 Rabbit 20.06 0.10 1.65 2.84 29 Rabbit 3 0.45 0.77 17.32 29.86 39 Rabbit 4 0.030.06 6.31 10.88 188 Rabbit 5 0.03 0.05 4.44 7.66 144 Mean 0.13 ± 0.180.23 ± 0.31 6.91 ± 6.06 11.91 ± 10.45 90 ± 72

Data presented as mean+SD.

Intravitreal delivery of DXR-PSA-PEG₃ micro or nanoparticles to rabbiteyes resulted in long-term drug release, sustained for at least 115 or105 days, respectively, the duration of the study. The released druglevels measured in the vitreous where much higher than those measured inthe aqueous humor, an average of 77-90-fold higher.

These data demonstrate sustained release from DXR-PSA-PEG₃ whendelivered intraocularly and suggest that DXR-PSA-PEG₃ will be apromising therapy for the treatment of NV ocular diseases including NVAMD.

Example 4. Synthesis and In Vitro Evaluation of Fully BiodegradableDXR-PSA-PEG₃ Rods

Rod-shaped DXR-PSA-PEG₃ conjugates were successfully produced with adiameter of 0.5 mm, a length of 0.5 cm, and a mass of 1 mg, with threedoxorubicin (DXR) drug loading levels, 10%, 30%, and 50%. DXR release invitro demonstrated release sustained for at least 25 days with all threerod types.

Materials and Methods

Preparation of PEG₃-PSA Polymer

(Polyethylene glycol)3-co-poly(sebacic acid)(PEG₃-PSA) was synthesizedby melt condensation. Briefly, sebacic acid was refluxed in aceticanhydride to form sebacic acid prepolymer (Acyl-SA). Polyethylene glycol(PEG₃) was prepared by mixing CH₃O-PEG-NH₂ (2.0 g), citric acid (26 g),dicyclohexylcarbodiimide (DCC; 83 mg) and 4-(dimethylamino)pyridine(DMAP, 4.0 mg) which were added to 10 mL methylene chloride, stirredovernight at room temperature, then precipitated and washed with ether,and dried under vacuum. Next, Acyl-SA (90% w/w) and PEG₃ (10% w/w) werepolymerized at 180° C. for 30 minutes. Nitrogen gas was swept into theflask for 30 seconds every 15 minutes.

Polymers were cooled to ambient temperature, dissolved in chloroform andprecipitated into excess petroleum ether. The precipitate was collectedby filtration and dried under vacuum to constant weight, to produce thePEG₃-PSA polymer.

Preparation of DXR-PSA-PEG₃ Rods

To prepare DXR-PSA-PEG₃ rods, three different concentrations of DXR wereused to produce rods with drug loading levels of 10%, 30% and 50% (w/w).For the 10%, 30% and 50% drug loaded rods, PEG₃-PSA and doxorubicinhydrochloride (DXR) (NetQem LLC, Durham, N.C.) were added to CHCl₃ atratios of 9:1, 7:3, and 1:1 (w/w). The PEG₃-PSA and DXR were incubatedat 50° C. for one hour after which the CHCl₃ was removed by vacuum. Thereaction product was grated to a fine powder and then compressed into aglass tube, with a diameter of 0.5 mm, which was used as a mold. Therods were extruded from the mold and cut to 0.5 cm lengths. Each rodweighed approximately 1 mg (0.9-1.2 mg).

In Vitro Drug Release

One rod (˜1 mg) was added to 1 ml of phosphate buffered saline (PBS, pH7.4) and incubated at 37° C. on a rotating platform (140 RPM). Atselected time points, supernatant was collected and fresh PBS added.DXR-conjugate concentration was measured by absorbance at 480 nm.

Results

Rod-shaped DXR-PSA-PEG₃ conjugates were produced with three differentdrug loading levels, 10%, 30%, and 50%. The DXR-PSA-PEG₃ conjugates wereformed into rods with a diameter of 0.5 mm, a length of 0.5 cm, and amass of 1 mg.

The duration of in vitro drug release was evaluated using theDXR-PSA-PEG₃ rods, with drug loading levels of 10%, 30%, and 50% (FIG. 8). Drug release from all three rods was sustained for at least 25 days.

These data demonstrate that the synthesis of rod-shaped DXR-PSA-PEG₃conjugates is possible. Rods composed of DXR-PSA-PEG₃ conjugates withdifferent drug concentrations were successfully synthesized, and allrods displayed sustained drug release in vitro. These data also suggestthat rods of differing sizes, mass, and drug content can be produced anddrug release rates optimized to obtain the most efficacious drugdelivery profile for each delivered drug and for each therapeuticindication.

Example 5. Production of DXR-PCPH-PSA-PEG₃ Polymer Conjugates

Microparticles composed of a fully biodegradable DXR-PSA-PCPH-PEG₃polymer drug conjugate were synthesized and displayed a slower drugrelease rate and more sustained drug release duration compared to theDXR-PSA-PEG₃ microparticles. The addition of PCPH to the polymerincreased the hydrophobicity of polymer-drug conjugate which resulted ina prolonged the duration of drug release, presumably due to a reductionin DXR solubility.

Materials and Methods

Synthesis of 1,6-bis(p-carboxyphenoxy)hexane (CPH)

1,6-bis(p-carboxyphenoxy)hexane (CPH) was synthesized as described byConix (1966). Briefly, p-hydroxybenzoic acid (27.6 g) and sodiumhydroxide (16.0 g) in water (80 mL) were stirred and heated to refluxtemperature. 1,6-dibromohexane (96%, 15.7 mL) was added over a period of30 min while maintaining at reflux temperature and refluxed for anadditional 3.5 hours. Sodium hydroxide (4.0 g) dissolved in water (10mL) was added to the mixture and refluxed for another 2 hours beforeallowing the reaction mixture to stand overnight at room temperature.The disodium salt was isolated by filtration, washed with 40 mL ofmethanol, and dissolved in distilled water. The solution was warmed to60-70° C. and acidified with 6 N sulfuric acid. The dibasic acid wasisolated by filtration and dried to constant weight under vacuum.

Synthesis of PreCPH

1,6-bis(p-carboxyphenoxy)hexane (CPH) (10.0 g) was refluxed in 200 mL ofacetic anhydride for 30 min under nitrogen, followed by removal ofunreacted diacid by filtration and solvent by evaporation. The residuewas recrystallized from dimethylformamide and ethyl ether, washed withdry ethyl ether, and dried to constant weight under vacuum.

Synthesis of PEG₃-PSA-PCPH Prepolymer

(Polyethylene glycol)₃-co-poly(sebacic acid)co-poly(CPH) (PEG₃-SA-PCPH)was synthesized by melt condensation. Briefly, sebacic acid was refluxedin acetic anhydride to form sebacic acid prepolymer (Acyl-SA).Polyethylene glycol (PEG₃) was prepared by mixing CH₃O-PEG-NH₂ (2.0 g),citric acid (26 g), dicyclohexylcarbodiimide (DCC; 83 mg) and4-(dimethylamino)pyridine (DMAP, 4.0 mg) which were added to 10 mLmethylene chloride, stirred overnight at room temperature, thenprecipitated and washed with ether, and dried under vacuum. Next, PEG₃(10% w/v), acyl-SA (60% w/v), and preCPH (30% w/v), were polymerized at180° C. for 30 minutes. Nitrogen gas was swept into the flask for 30seconds every 15 minutes. Polymers were cooled to ambient temperature,dissolved in chloroform and precipitated into excess petroleum ether.The precipitate was collected by filtration and dried under vacuum toconstant weight, to produce the PEG₃-PSA-PCPH prepolymer.

Preparation of DXR-PSA-PCPH-PEG₃ Microparticles

To prepare DXR-PSA-PCPH-PEG₃ microparticles, 200 mg PEG₃-PSA-PCPH wasdissolved in 3 mL dichloromethane (DCM) and mixed with 40 mg doxorubicinhydrochloride (DXR) (NetQem LLC, Durham, N.C.) dissolved in 1.5 mL DMSO.Following incubation at 50° C. for 30 min, the mixture was homogenizedin 100 mL of PVA at 3,000 rpm for 1 min. After stirring for 2 hr,particles were collected by centrifugation (9,000×g for 25 minutes) andwashed thrice before lyophilization.

Particle Characterization

Particle size was determined using a Coulter Multisizer IV(Beckman-Coulter Inc., Fullerton, Calif.). Greater than 100,000particles were sized for each batch of microparticles to determine themean particle diameter.

In Vitro Drug Release

DXR-PSA-PCPH-PEG₃ microparticles (2 mg) were suspended in phosphatebuffered saline (PBS, pH 7.4), and incubated at 37° C. on a rotatingplatform (140 RPM). At selected time points, supernatant was collectedby centrifugation (13,500×g for 5 min) and particles were resuspended infresh PBS. DXR-conjugate was measured by absorbance at 480 nm.

Results

DXR-PSA-PCPH-PEG₃ microparticles were synthesized and displayed a meansize of 24.3±8.7 um with a drug loading level of 13.9%.

The duration of in vitro drug release was compared between theDXR-PSA-PCPH-PEG₃ microparticles and the DXR-PSA-PEG₃ microparticles(mean size 22.5+8.3 um). The DXR-PSA-PEG₃ microparticles showed drugrelease sustained for 45 days while the DXR-PSA-PCPH-PEG₃ microparticlesdemonstrated a slower drug release rate and drug release sustained forover 75 days (FIG. 9 ).

Microparticles composed of a fully biodegradable DXR-PSA-PCPH-PEG₃polymer drug conjugate were synthesized. The DXR-PSA-PCPH-PEG₃microparticles displayed a slower drug release rate and more sustaineddrug release duration compared to the DXR-PSA-PEG₃-microparticles,particles lacking the addition of the CPH polymer. The addition of CPHto the polymer increased the hydrophobicity of the released drugconjugate which is expected to reduce the solubility of DXR, andresulted in a prolonged duration of drug release.

These data demonstrate that by altering the polymer chemistry toincrease the hydrophobicity of the released drug conjugate, the leveland duration of drug release can be modified, indicating that theseparameters can be optimized to obtain the most efficacious drug deliveryprofile for each delivered drug and for each therapeutic indication.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A polymer conjugate of the formula

wherein A is an ophthalmic drug for treatment of the posterior segmentof the eye selected from brimonidine, apraclonidine, brinzolamide,acetazolamine, dorzolamide, timolol, carteolol, betaxolol, andlevobetaxolol; Z is a hydrophilic polymer segment wherein the segment ispoly(ethylene glycol); Y is a multivalent branch point that is anorganic, inorganic, or organometallic moiety; X is a hydrophobic polymersegment wherein the segment is selected from a poly(lactic acid)segment, a poly(glycolic acid) segment, and a poly(lactic-co-glycolicacid) segment; and m and n are independently selected from 1, 2, 3, 4,5, 6, 7, 8, 9, or
 10. 2. The polymer conjugate of claim 1, wherein Y isselected from


3. The polymer conjugate of claim 1, wherein X is a poly(lactic acid)segment.
 4. The polymer conjugate of claim 1, wherein A is timolol. 5.The polymer conjugate of claim 1, wherein A is carteolol.
 6. The polymerconjugate of claim 1, wherein A is betaxolol.
 7. The polymer conjugateof claim 1, wherein A is levobetaxolol.
 8. The polymer conjugate ofclaim 1, wherein X is a poly(glycolic acid) segment.
 9. The polymerconjugate of claim 8, wherein A is timolol.
 10. The polymer conjugate ofclaim 8, wherein A is carteolol.
 11. The polymer conjugate of claim 8,wherein A is betaxolol.
 12. The polymer conjugate of claim 8, wherein Ais levobetaxolol.
 13. The polymer conjugate of claim 1, wherein X is apoly(lactic-co-glycolic acid) segment.
 14. The polymer conjugate ofclaim 13, wherein A is timolol.
 15. The polymer conjugate of claim 13,wherein A is carteolol.
 16. The polymer conjugate of claim 13, wherein Ais betaxolol.
 17. The polymer conjugate of claim 13, wherein A islevobetaxolol.
 18. A pharmaceutical composition containingmicroparticles comprising the polymer conjugate of claim 1 and apharmaceutically acceptable excipient.
 19. A method of treating adisease or disorder of the eye selected from wet-age related maculardegeneration, choroidal neovascularization, glaucoma, cornealneovascularization, and retinal neovascularization comprisingadministering to a patient in need thereof the pharmaceuticalcomposition of claim
 18. 20. The method of claim 19, wherein thepharmaceutical composition is administered via intravitreal injection,subconjunctival injection, intracameral injection, intrastromalinjection, intracorneal injection, subretinal injection, or intraocularinjection.